Methods and compositions of targeted drug development

ABSTRACT

Provided herein are compounds having anti-proliferative effect. Also provided are compounds that can modulate the activity of multi-domain proteins comprising a dimerization arm and interdomain tether, such as EGFR, where an untethered, extended conformation is the active state and a tethered conformation is the inactive state, resulting in an autoinhibited configuration. Also provided are methods and pharmacophores for identifying such compounds. Other aspects provide methods or therapeutic treatment for proliferative diseases, disorders, or conditions, such as those associated with EGFR.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Nonprovisional application Ser. No. 13/521,010 filed 6 Jul. 2012 (Int'l. filing date 6 Jan. 2011); which claims the benefit of priority to Int'l. Application No. PCT/US11/20418 filed 6 Jan. 2011 and U.S. Provisional Application No. 61/292,776, filed 6 Jan. 2010; each of which is incorporated herein by reference in its entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The Sequence Listing, which is a part of the present disclosure, includes a computer readable form comprising nucleotide and/or amino acid sequences of the present invention. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to novel chemical compounds for use in the treatment of disease, methods of manufacture of such compounds and intermediates thereof, and methods of identifying lead molecules for use in quasi-rational drug design.

BACKGROUND OF THE INVENTION

Rational drug development is a process of developing lead molecules, not by randomly screening thousands of molecules in the blind hope of finding one that shows the desired activity, but rather by deducing the active site of the target and devising a chemical that interacts with that site in the appropriate manner.

Epidermal Growth Factor Receptor (EGFR) is a member of the ErbB (HER) family receptor tyrosine kinase (RTKs), which regulate cell growth and differentiation and are implicated in many human cancers. EGFR activation and dimerization is discussed in, for example, Burgess et al. (2003) Moledular Cell 12, 541-552 and Ferguson et al. (2003) Molecular Cell 11, 507-517.

EGF activates its receptor by inducing dimerization of the extracellular region of EGFR. The activation of EGFR has been described through results of disulfide bond mapping as well as X-ray crystal structures. The crystal structures of ligand-bound sEGFR showed that dimerization is receptor mediated, with two individual ligand molecules present in the dimer. The dimerization interface of activated EGFR is completely occluded by intramolecular interactions, and is an autoinhibited configuration. To activate the receptor, a large domain rearrangement that exposes this occluded interface must accompany EGF binding where EGF does not contribute to the EGFR dimer interface. The EGFR mechanism is in sharp contrast to most other receptor tyrosine kinase activation mechanisms in which the bound ligand contributes directly to the receptor dimerization interface and does not dramatically alter the conformation of the extracellular region of the receptor tyrosine kinase.

EGFR contains four subdomain I, II, III, and IV. Almost all receptor/receptor contacts observed in the crystal structures of EGFR are mediated by domain II. At the center of the dimer interface is a prominent loop (residues 242-259 of EGFR) that extends from the second C1 module (module 5) of each domain II and reaches across the interface to interact primarily with domain II of its dimerization partner. This domain II loop, which is specific to ErbB receptors, is the “dimerization arm”. The dimerization arm of domain II is completely occluded by intramolecular interactions with domain IV (i.e., an autoinhibited configuration). There are two smaller interaction sites in the dimer that involve side chains from the second and the sixth disulfide-bonded modules of domain II. And the dimer interface may extend into domain IV. While the two receptor molecules approach one another very closely toward the C terminus of domain IV, a well-defined, tight interface is not observed.

Although EGF and TGF-α clearly do not span the dimer interface, each ligand simultaneously contacts two separate binding surfaces in the same EGFR molecule. The bound EGF or TGF-α molecule resembles a wedge between domains I and III. The relationship between domains I and II is essentially identical to that seen in IGF-1R and in the activated sEGFR dimer, implying that ligand binding does not greatly influence the relative orientation of these two domains. But the relationship between domains II and III differs dramatically in the activated and unactivated structures. A direct intramolecular interaction between cysteine-rich domains II and IV restrains the domain II/III relationship that characterizes the unactivated configuration. This interdomain “tether” is stabilized by essentially identical interactions between the two cysteine rich domains (II and IV) in inactive sErbB3 and sEGFR.

The intramolecular domain II/IV tether precisely buries the dimerization arm of domain II against domain IV, so that the tethered configurations of sErbB3 and sEGFR cannot dimerize and thus appear to be autoinhibited. Moreover, the two ligand binding surfaces on domain I and III are too far apart in the tethered configuration for a single ligand to bind to both simultaneously. Consequently, the tethered configuration can only form low-affinity interactions with ligand, using just one of its ligand binding surfaces at a time.

Switching between the unactivated and activated configurations of sEGFR requires domains I and III to be drawn toward one another through a 130° rotation of the rigid domain I/II pair in one plane and a 20° rotation in another. Only this extended configuration of sEGFR is capable of both high-affinity ligand binding and efficient dimerization.

Based upon energetic calculations, it is currently thought that at any given time, about 95% of sEGFR molecules will be tethered and the remaining 5% will not. The presence of ligand and subsequent binding to domains I and III of the non-tethered form will drive the equilibrium toward the non-tethered form, trapping receptor molecules in the extended state that can dimerize.

Exposure of the dimerization arm is not sufficient alone to drive EGFR dimerization. Also required is additional contact sites in modules 2 and 6 of domain III. These two additional contact sites and the dimerization arm cooperate at the dimer interface.

Known strategies of EGFR inhibition are directed to antibody binding of domain III to provide steric hindrance of the required configuration change (e.g., Erbitux). Other conventional strategies are directed to antibody binding of domain II, specifically the dimerization arm, so as to prevent dimerization (e.g., pertuzumab). Still other conventional strategies are directed to antibody binding of domain IV residues that participate in the intramolecular tether (e.g., trastuzumab, Herceptin). But no existing strategies are directed to the tethering mechanism of activation.

SUMMARY OF THE INVENTION

Described herein are compounds and compositions having an anti-proliferative effect, along with methods of therapeutic treatment with such compounds. Also provided are methods of discovery of such compounds. An approach described herein identifies modulators of the activity of multi-domain proteins comprising a dimerization arm and interdomain tether, such as EGFR, where an untethered, extended conformation is the active state and a tethered conformation is the inactive state, resulting in an autoinhibited configuration.

One aspect of the invention provides small molecule compounds.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, a 1734-like compound inhibits EGFR activity and comprises at least five or more of functional groups F(II)1, F(II)2, F(II)3, F(II)4, F(II)5, F(II)6, F(II)7, F(II)8, and F(II)9 of a Scheme II 1734-like pharmacophore, with functional groups as defined herein.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, a 1886-like compound inhibits EGFR activity and comprises at least seven or more of functional groups F(III)1, F(III)2, F(III)3, F(III)4, F(III)5, F(III)6, F(III)7, F(III)8, and F(III)9 of a Scheme III 1886-like pharmacophore, with functional groups as defined herein.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, a 10381-like compound inhibits EGFR activity and comprises at least eight or more of functional groups F(IV)1, F(IV)2, F(IV)3, F(IV)4, F(IV)5, F(IV)6, F(IV)7, F(IV)8, F(IV)9, and F(IV)10 of a Scheme IV 10381-like pharmacophore, with functional groups as defined herein.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, compounds have a formula of

with constituents as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, a 11091-like compound inhibits EGFR activity and comprises at least nine or more of functional groups F(V)1, F(V)2, F(V)3, F(V)4, F(V)5, F(V)6, F(V)7, F(V)8, F(V)9, F(V)10, and F(V)11 of a Scheme V 11091-like pharmacophore, with functional groups as defined herein.

One aspect of the invention provides a method of treating a proliferative disease, disorder, or condition using a compound described herein.

In one embodiment, the method of treating a proliferative disease, disorder, or condition comprises administering to a subject in need thereof a composition comprising a therapeutically effective amount of a compound with a formula selected from the group consisting of: Formula (13); Formula (14); Formula (15); Formula (16); Formula (18); Formula (19); Formula (20); Formula (21); Formula (22); Formula (23); Formula (25); Formula (27); Formula (28); Formula (32); and Formula (33), as defined herein, or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, the method of treating a proliferative disease, disorder, or condition comprises administering to a subject in need thereof a composition comprising a therapeutically effective amount of a compound with a formula selected from the group consisting of: AD4-1734 (Formula (12)); AD4-1886 (Formula (17)); AD4-10381 (Formula (24)); and AD4-11091 (Formula (31)), or a stereoisomer or pharmaceutically acceptable salt thereof.

In one embodiment, the method of treating a proliferative disease, disorder, or condition comprises administering to a subject in need thereof a composition comprising a therapeutically effective amount of a compound with a formula selected from the group consisting of:

or a stereoisomer or pharmaceutically acceptable salt thereof.

In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient.

In some embodiments, the treated proliferative disease, disorder, or condition is selected from: cancer; a blood vessel proliferative disorder; a fibrotic disorder; a mesangial cell proliferative disorder; psoriasis; actinic keratoses; seborrheic keratoses; warts; keloid scars; eczema; and hyperproliferative diseases caused by a viral infection.

In some embodiments, the treated proliferative disease, disorder, or condition is associated with EGFR. In some configurations, the proliferative disease, disorder, or condition associated with EGFR is selected from the group consisting of: cancer; a blood vessel proliferative disorder; a fibrotic disorder; a mesangial cell proliferative disorder; psoriasis; actinic keratoses; seborrheic keratoses; warts; keloid scars; eczema; and hyperproliferative diseases caused by a viral infection.

One aspect provides a method for identifying an EGFR inhibitor.

In one embodiment, the method for identifying an EGFR inhibitor comprises: providing a pharmacophore comprising a scheme selected from the group consisting of Scheme II (as defined herein) as input to a 3-dimensional database; comparing a three dimensional structure of a candidate compound to the three dimensional structure of the pharmacophore; and selecting a candidate compound with a three dimensional structure that substantially aligns with five or more functional groups of Scheme II (ADS-1734-like).

In one embodiment, the method for identifying an EGFR inhibitor comprises: providing a pharmacophore comprising a scheme selected from the group consisting of Scheme III (as defined herein) as input to a 3-dimensional database; comparing a three dimensional structure of a candidate compound to the three dimensional structure of the pharmacophore; and selecting a candidate compound with a three dimensional structure that substantially aligns with seven or more functional groups of Scheme III (AD4-1886-like).

In one embodiment, the method for identifying an EGFR inhibitor comprises: providing a pharmacophore comprising a scheme selected from the group consisting of Scheme IV (as defined herein) as input to a 3-dimensional database; comparing a three dimensional structure of a candidate compound to the three dimensional structure of the pharmacophore; and selecting a candidate compound with a three dimensional structure that substantially aligns with eight or more of functional groups of Scheme IV (AD4-10381-like).

In one embodiment, the method for identifying an EGFR inhibitor comprises: providing a pharmacophore comprising a scheme selected from the group consisting of Scheme V (as defined herein) as input to a 3-dimensional database; comparing a three dimensional structure of a candidate compound to the three dimensional structure of the pharmacophore; and selecting a candidate compound with a three dimensional structure that substantially aligns with nine or more of functional groups of Scheme V (AD4-11091-like).

In some embodiments of the method for identifying an EGFR inhibitor, similarity between the three-dimensional structure of the candidate compound and the three-dimensional structure of the pharmacophore is indicative of an ability of the candidate compound to inhibit EGFR by substantially maintaining a tethered inactive configuration of EGFR or substantially preventing stabilization of the untethered active configuration of EGFR.

Some embodiments of the method for identifying an EGFR inhibitor further comprise determining identity and spatial orientation of at least a portion of atoms of EGFR associated with stabilizing a tethered configuration of domain II and domain IV of EGFR in an inactive conformation; and constructing a pharmacophore, wherein the pharmacophore comprises a plurality of pharmacophoric features that approximates the identity and the spatial orientation of the at least a portion of atoms of EGFR associated with stabilizing a tethered configuration of domain II and domain IV of EGFR in an inactive conformation such that the pharmacophore structural features are complementary to the inactive EGFR configuration. In some configurations, determining identity and spatial orientation of at least a portion of atoms of EGFR associated with stabilizing a tethered configuration of domain II and domain IV of EGFR in an inactive conformation comprises analysis of X-ray crystallographic data derived from a crystalline form of EGFR in an inactive, tethered conformation.

In some embodiments of the method for identifying an EGFR inhibitor, at least one pharmacophoric feature approximates identity and spatial orientations of at least a portion of atoms of domain II of EGFR in a tethered inactive conformation. In some embodiments of the method for identifying an EGFR inhibitor, at least one pharmacophoric feature approximates identity and spatial orientations of at least a portion of atoms of a cleft region between domain II and domain IV of EGFR in a tethered inactive conformation.

Some embodiments of the method for identifying an EGFR inhibitor further comprise determining a docking affinity of the candidate molecule for the at least a portion of atoms of EGFR associated with stabilizing a tethered configuration of domain II and domain IV of EGFR in an inactive conformation. In some configurations, docking affinity is quantified by energy gained upon interaction of the candidate molecule with the target biomolecule, energy required to attain the docked conformation relative to the lowest energy conformation, or a combination thereof.

Other objects and features will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1A shows pharmacophore 21_thr100_glu105 (see Example 3; Table 1). FIG. F10A: Pharmacophore 21_thr100_glu105. FIG. F10B: Pharmacophore 21_thr100_glu105 aligned to AD4-1886.

FIG. 1B shows AD4-1886 aligned to pharmacophore 21_thr100_glu105.

FIG. 2: AD4-11091 aligned to the pharmacophore model, Pharm 1886-6. (see Example 3; Table 3).

FIG. 3 shows the binding site at the interface of Domain II and Domain IV of the Inactive Form of EGFr (1NQL.pdb) as determined by the site finder in MOE. The Carbon Atoms of the Domain II Residues are Colored Red and those of the Domain IV Residues are Colored Blue.

FIG. 4 shows Pharm-1 nql-glue-6 aligned to the hit AD4-1734.

FIG. 5 shows DockPharm 1505-2 aligned to the hit AD4-10381.

FIG. 6A-FIG. 6F are a series of two-dimensional representations of AD4-1886 and AD4-1886-like compounds docked with the EGFR/cetuximab complex. Docking of compound AD4-1886 to EGFR is depicted, for example, in FIG. 6A. Docking of compound AD4-11883 (an AD4-1886-like compound) to EGFR is depicted, for example, in FIG. 6B. Docking of compound AD4-11975 (an AD4-1886-like compound) to EGFR is depicted, for example, in FIG. 6C. Docking of compound AD4-11409 (an AD4-1886-like compound) to EGFR is depicted, for example, in FIG. 6D. Docking of compound AD4-11638 (an AD4-1886-like compound) to EGFR is depicted, for example, in FIG. 6E. Docking of compound AD4-11645 (an AD4-1886-like compound) to EGFR is depicted, for example, in FIG. 6F.

FIG. 7A-FIG. 7F are a series of two-dimensional representations of AD4-11091 and AD4-11091-like compounds docked with the EGFR/cetuximab complex. Docking of compound AD4-11091 to EGFR is depicted, for example, in FIG. 7A. Docking of compound AD4-12509 (an AD4-11091-like compound) to EGFR is depicted, for example, in FIG. 7B. Docking of compound AD4-12423 (an AD4-11091-like compound) to EGFR is depicted, for example, in FIG. 7C. Docking of compound AD4-12528 (an AD4-11091-like compound) to EGFR is depicted, for example, in FIG. 7D. Docking of compound AD4-12522 (an AD4-11091-like compound) to EGFR is depicted, for example, in FIG. 7E. Docking of compound AD4-12504 (an AD4-11091-like compound) to EGFR is depicted, for example, in FIG. 7F.

FIG. 8A-FIG. 8F are a series of two-dimensional representations of AD4-1734 and AD4-1734-like compounds docked with inactive EGFR. Docking of compound AD4-1734 to EGFR is depicted, for example, in FIG. 8A. Docking of compound AD4-10631 (an AD4-1734-like compound) to EGFR is depicted, for example, in FIG. 8B. Docking of compound AD4-10188 (an AD4-1734-like compound) to EGFR is depicted, for example, in FIG. 8C. Docking of compound AD4-10186 (an AD4-1734-like compound) to EGFR is depicted, for example, in FIG. 8D. Docking of compound AD4-10633 (an AD4-1734-like compound) to EGFR is depicted, for example, in FIG. 8E. Docking of compound AD4-10174 (an AD4-1734-like compound) to EGFR is depicted, for example, in FIG. 8F.

FIG. 9A-FIG. 9F are a series of two-dimensional representations of AD4-10381 and AD4-10381-like compounds docked with inactive EGFR. Docking of compound AD4-10381 to EGFR is depicted, for example, in FIG. 9A. Docking of compound AD4-11340 (an AD4-10381-like compound) to EGFR is depicted, for example, in FIG. 9B. Docking of compound AD4-12632 (an AD4-10381-like compound) to EGFR is depicted, for example, in FIG. 9C. Docking of compound AD4-12681 (an AD4-10381-like compound) to EGFR is depicted, for example, in FIG. 9D. Docking of compound AD4-12732 (an AD4-10381-like compound) to EGFR is depicted, for example, in FIG. 9E. Docking of compound AD4-11511 (an AD4-10381-like compound) to EGFR is depicted, for example, in FIG. 9F.

FIG. 10A-FIG. 10B are a series of line and scatter plots showing % inhibition of EGFR as a function of concentration of Tykerb (FIG. 10A) or Iressa (FIG. 10B) either alone or in combination with AD4-10628. A shift in the dose-response curve to the left indicates a more potent response.

FIG. 11 is a scatter plot showing Dose Reduction Index (DRI) as a function of Fa for DRI Tykerb and DRI AD4-10628.

FIG. 12 is a histogram showing a summary of Combination Index (CI) values at 90% inhibition (ED90) for a series of AD4 compounds in combination with Tykerb. Compound 4 is Iressa. Compound 5 is Tarceva. The balance of compounds are AD4 compounds described herein. Response below the dark middle line (i.e., CI<0.9) indicates synergism.

FIG. 13A-FIG. 13D are a cartoon depicting conformations of EGFR. FIG. 13A shows EGFR as a tethered monomer. FIG. 13B shows EGFR as an untethered monomer. FIG. 13C shows EGFR in a ligand stabilized extended conformation. FIG. 13D shows EGFR as a ligand induced activated dimer.

FIG. 14 is a cartoon depicting ligand-induced dimerization and activation of the kinase domain of EGFR.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

Described herein are compounds and compositions having an anti-proliferative effect, along with methods of therapeutic treatment with such compounds and methods of discovery of such compounds. Various small molecule compounds described herein can hold proteins of multiple domains together in a tethered, inactive state. Also provided are methods to identify the structural requirements of such inhibitors, screen for effective inhibitors, optimize the structure of identified candidates, and utilize identified small molecule compounds in therapeutic treatment regimes.

One aspect of the invention is directed to small molecule compounds efficacious in treating proliferative diseases or conditions. Various embodiments of compounds described herein can have an anti-proliferative effect. Various embodiments of compounds described herein can hold multiple domain proteins in a tethered, inactive state. Various embodiments of compounds described herein can have an inhibitory effect on EGFR. Compounds described herein have been demonstrated to be empirically effective in treating proliferative diseases and conditions.

One aspect of the invention is directed to therapeutic treatment of proliferative diseases and disorders using compounds and compositions described herein.

One aspect of the invention is directed to compounds, methods, and apparatuses for developing one or more drugs for one or more targeted therapies. More specifically, the approach described herein identifies modulators of the activity of multi-domain proteins comprising a dimerization arm and interdomain tether, where an untethered, extended conformation is the active state and a tethered conformation is the inactive state, resulting in an autoinhibited configuration. The pharmacophoric approach described herein is based upon a mechanistic understanding of conformation-dependent protein receptor activation mechanisms, thus avoiding conventional combinatorial chemistry and high throughput screening techniques.

Biomolecule Target Selection

Desirable target enzymes include those for which there exists crystallography data sufficient to discern a ligand binding, activation, and/or dimerization mechanism. The various methods of the invention can be used to generate pharmacophore models for a variety of multi-domain protein targets (crystallized with and/or without ligand) having an interdomain tether associated with activation state. Thus is provided compounds that can prevent untethering and stabilization of the extended conformation, and methods for identifying such compounds.

It shall be understood that the types of biomolecule target for the lead molecules generated by the methods of the present invention can include one or more of EGFR (i.e., ErbB1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4).

EGFR

Described herein is targeting of various portions of the domains of EGFR so as to prevent stabilization of the untethered, extended conformation. In other words, a small molecule inhibitor can be used to hold proteins of domain II and IV together in the tethered, inactive state. This strategy can provide for some retention of the basal levels of EGFR signaling, retention of EGF response, and/or reduce EGF-independent dimerization. Such a therapeutic effect would slow rapid growth of cancer cells (which are more sensitive given increased expression levels of EGFR) but retain at least a portion of basal EGFR activity necessary for healthy tissue function.

Known strategies of EGFR inhibition are directed to antibody binding of domain III to provide steric hindrance of the required configuration change (e.g., Erbitux). Other conventional strategies are directed to antibody binding of domain II, specifically the dimerization arm, so as to prevent dimerization (e.g., pertuzumab). Still other conventional strategies are directed to antibody binding of domain IV residues that participate in the intramolecular tether (e.g., trastuzumab, Herceptin). But, in contrast to the approach described herein, the above conventional strategies do not prevent untethering or stabilization of the extended conformation.

As described above, EGFR has an autoinhibited configuration in which the dimerization arm of domain II is completely occluded by intramolecular interactions with domain IV (see e.g., FIG. 13A). EGF activates its receptor by inducing dimerization of the extracellular region of EGFR (see e.g., FIG. 13D). Almost all receptor/receptor contacts observed in the crystal structures of EGFR are mediated by domain II, specifically, a prominent loop (residues 242-259 of EGFR) that extends from the second C1 module (module 5) of each domain II (i.e., the dimerization arm). The unactivated configuration is characterized by a direct intramolecular interaction between cysteine-rich domains II and IV, which restrains the domain II/III relationship (see e.g., FIG. 13A). This interdomain “tether” is stabilized by essentially identical interactions between the two cysteine rich domains (II and IV) in inactive sEGFR. Switching between the unactivated and activated configurations of sEGFR requires domains I and III to be drawn toward one another through a 130° rotation of the rigid domain I/II pair in one plane and a 20° rotation in another (see e.g., FIG. 13B). Only this extended configuration of sEGFR is capable of both high-affinity ligand binding (see e.g., FIG. 13C) and efficient dimerization (see e.g., FIG. 13D). In the activated and dimerized configuration, the dimerization arm of domain II reaches across the interface to interact primarily with the corresponding domain II arm of its dimerization partner (see e.g., FIG. 13D). EGFR dimerization also requires interaction of contact sites in modules 2 and 6 of domain III. The presence of EGF ligand and subsequent binding to domains I and III of the non-tethered form will drive the equilibrium toward the non-tethered form, trapping receptor molecules in the extended state that can dimerize.

The approach described herein provides for some retention of the basal levels of EGFR signaling. In healthy individuals there exists a baseline signal from EGFR necessary for growth, with enhanced EGF levels promoting accelerated growth in, for example, wound recovery. But cancer cells have been demonstrated to exhibit more EGFR, which increases the probability of the untethered conformation and subsequent EGF binding to the unoccluded domain I/III ligand binding site, thereby activating EGFR. An inhibitor that holds multi-domain proteins of EGFR together in a tethered, inactive state can allow for some basal levels of EGFR signaling.

Furthermore, small molecule inhibitors that prevent stabilization of the untethered state (see e.g., FIG. 14B depicting untethered state, and FIG. 14C depicting stabilized untethered state) of EGFR can be used in conjunction with other anti-EGFR therapeutic agents. Use of small molecule inhibitors described herein in conjunction with other anti-EGFR therapeutic modalities can allow decreased dosage and/or increased maximal inhibition. For example, use of such small molecule inhibitors can be used in conjunction with Erbitux (which binds domain III blocking EGF), which would allow a lower dosage of Erbitux and/or increased maximal inhibition.

In various embodiments, domain II of EGFR, as existing in the tethered state, is targeted so as to prevent opening (i.e., the configuration change from tethered to open, see e.g., FIG. 13A-B depicting tethered and untethered conformations). In various embodiments, the cleft between domain II and domain IV is targeted so as to prevent opening (i.e., the configuration change from tethered to open). A single small molecule can be used to span the two domains. Alternatively, a series of small molecules (e.g., at least two small molecules) in several compartments can be used in conjunction so as to span the two domains. In various embodiments, domain II of EGFR, as existing in the untethered state, is targeted so as to prevent stabilization of the untethered state (see e.g., FIG. 13B-C, depicting untethered and stabilized conformations). In various embodiments, domain III of EGFR is targeted (e.g., modules 2 and 6 of domain III, which are interacting contact sites required for EGFR dimerization) so as to prevent stabilization of the untethered state.

Pharmacophoric Approach

One aspect is directed to a pharmacophoric approach for developing a drug targeting a multi-domain protein having an interdomain tether associated with activation state. Based upon the activation and dimerization mechanism of a biomolecule of interest, binding targets are identified and characterized. The mechanism and/or binding target can be characterized, for example, via crystallography data. The target binding domains can be expressed as one or more pharmacophore features and/or compiled in a pharmacophore model comprising one or more pharmacophore features.

Pharmacophore generation can be according to software designed for such a task. Candidate molecules (from, for example, one or more chemical libraries) can be selected from those molecules which align to the pharmacophore models. Preferably, candidate molecules are docked and scored in silico for interaction with the target binding domain. Again, docking and scoring can be according to software designed for such a task. After selection of molecules aligning to one or more pharmacophore models, with optional docking and scoring in silico, the selected molecules can be obtained, for example, by chemical synthesis or from a commercial source. The selected molecules can be measured for binding affinity and/or effect on function for the target biomolecule. Such assessment can be according to a biological assay. The tested molecules can be further selected according to desirable measured parameters. The selected molecules and/or the further selected molecules can optionally be further optimized.

Determining Structure Spatial Position

From the activation and dimerization model of the target biomolecule, target regions can be identified and 3D binding domains can be defined. Definition of the binding domain(s) generally involves the determination of the specific spatial position of the atoms of the portion of the target biomolecule which plays a role in the activation and dimerization mechanism.

Determination of the spatial position of the binding portion can be achieved by means of various in silico techniques. For example, software packages can be used that model the structure of the binding surface and match it to a model of the active surface of the target to assess levels of compatibility. Such software includes CAMAL.

Determination of the spatial position of the binding portion can be achieved by means of X-ray crystallography. X-ray crystallography can be used to determine the structure of atoms within a structure that is known to play a role in the activation and dimerization mechanism, and to then use this structural information to build a synthetic molecule that binds to one or more of these components and interferes with configuration changes and/or stabilization. Techniques for employing X-ray crystallography for structural determination are known in the art (see e.g., Messerschmidt (2007) X-Ray Crystallography of Biomacromolecules: A Practical Guide, John Wiley & Sons, ISBN-10: 3527313966; Woolfson (2003) An Introduction to X-ray Crystallography, 2d Ed., Cambridge University Press, ISBN-10: 0521423597). Creation of X-ray crystal structures are also known in the art (see e.g., U.S. Pat. No. 6,931,325 to Wall and U.S. Pat. No. 6,916,455 to Segelke, each incorporated herein by reference). Except as otherwise noted herein, therefore, the process of the present invention can be carried out in accordance with such processes.

Parameters derived from X-ray crystallography observed diffraction data include, but are not limited to, hydrogen bonders, apolar hydrophobic contacts, salt bridge interactions, polar surface area of the domain, apolar surface area of the domain, shape complementarily score for the antibody-target complex, and explicitly placed water molecules. Also useful is characterization of bonds between atoms. The distance between two atoms that are singly bonded ranges from about 1.45 to about 1.55 Å. Atoms that are double bonded together are typically about 1.2 to about 1.25 Å apart. Bonds that are resonant between single and double bonds typically have an about 1.30 to about 1.35 Å separation.

Construction of Pharmacophores

A pharmacophore model can be constructed from structural information of biomolecule components playing a role in activation and dimerization, including definition of atom position. Small molecules with complementary features to components of the target biomolecule, such as a component playing a role in activation and dimerization, have the potential to interfere with configuration changes and/or stabilization necessary for activation and dimerization and thus have therapeutic utility.

In various embodiments, in silico approaches can be used for de novo structure design with a fragment based approach employing contact statistics, 3D surface models, and docked ligands as templates. From the spatial position information, and/or from other parameters described above, one can derive 3D ligand-receptor models (e.g., interaction pattern, pharmacophore schemes), surface maps (e.g., topography/shape, electrostatic profile, hydrophobicity, protein flexibility), and docking models (e.g., scoring system for ligand binding, minimum energy calculation).

Techniques for pharmacophore model construction are known in the art and described extensively herein (see e.g., Examples 4-5). Except as otherwise noted herein, therefore, the processes of the present invention can be carried out in accordance with such processes.

A pharmacophore model or scheme is generally a set of structural features in a ligand that are related, preferably directly related, to the ligand's recognition at a receptor site and its biological activity. Pharmacophore features can be derived from corresponding donor, acceptor, aromatic, hydrophobic, and/or acidic or basic moieties of the corresponding target biomolecule, especially those features on domains participating in dimerization and activation mechanisms. It shall be understood that additional information about the nature of the atoms in the target biomolecule being used in a pharmacophore scheme, and not simply the spatial location of the atoms, can assist in the modeling process of a new chemical lead. These characteristics include, but are not limited to, the pKa values of the atoms, the rotational rigidity of the bonds holding the atoms in place, the nature of the bonds themselves (single, double, resonant, or otherwise), the projected directionality of hydrogen bond donors and acceptors, etc.

Typical feature components useful in generating a pharmacophore scheme include, but are not limited to, atomic position; atomic radii; hydrogen bond donor features; hydrogen bond acceptor features; aromatic features; donor features; acceptor features; anion features; cation features; acceptor and anion features; donor and cation features; donor and acceptor features; acid and anion features; hydrophobic features, hydrogen bond directionality, and metal ligands (see e.g., Examples 4-5). Such features can be located, for example, at a single atom, centroids of atoms, or at a projected directional position in space.

It is contemplated that numerous pharmacophore queries can be designed for any given target biomolecule. It is further contemplated that these pharmacophore queries will be useful to identify small molecule ligands which interact with the target biomolecule at a site involved with dimerization and activation, especially towards maintaining a tethered, inactive conformation.

Exemplary resources for accomplishing such modeling and queries include, but are not limited to MOE (CGG) (providing pharmacophore query and visualization), Glide (Schrodinger) (providing docking and scoring), Accord for Excel (Accelrys) (providing organization of molecular information including chemical structures and formulas), and the ZINC database (UCSF) (providing a library of commercial compounds). One design tool for the generation of pharmacophores from immune system protein-target biomolecule structural binding characterization is MOE, or Molecular Operating Environment (Chemical Computing Group). Model generation uses geometrical and electronic constraints to determine the 3D positions of features corresponding to the immune system protein. The model of these embodiments consists of spherical features in 3D space. The diameter of the spheres can be adjusted (e.g., about 0.5 to about 3.0 Å). Such models allow matches and/or partial matches of the features.

Pharmacophoric structural features can be represented by labeled points in space. Each ligand can be assigned an annotation, which is a set of structural features that may contribute to the ligand's pharmacophore (see e.g., Examples 4-5). In various embodiments, a database of annotated ligands can be searched with a query that represents a pharmacophore hypothesis (see e.g., Examples 6-7). The result of such a search is a set of matches that align the pharmacophoric features of the query to the pharmacophoric features present in the ligands of the searched database (see e.g., Examples 6-7, Tables 11-15). The number of hits within the database depends, at least in part, upon the size of the database and the restrictiveness of the pharmacophore query (e.g., partial matches, number of features, etc.). Properties and parameters of the molecules present within the search database are used to focus the outcome of the query. For example, compounds with a defined range of molecular weight (MW) or lipophilicity (log P) can be present in the searched section of the library database of compounds.

Candidate Molecules

The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). As described above, candidate molecules can be searched using a pharmacophore query. Candidate molecules encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups for structural interaction with proteins, particularly hydrogen bonding, and can include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

In preferred embodiments, the candidate molecules are compounds in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example: ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals etc).

Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 D) with relatively fewer features (e.g., less than about 3 hydrogen donors and/or less than about 6 hydrogen acceptors; hydrophobicity character x log P of about −2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 D) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and/or less than about 8 rotatable bonds; hydrophobicity character x log P of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Preferably, initial screening is performed with lead-like compounds.

When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being “drug-like”. Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical success if it is drug-like.

Several of these “drug-like” characteristics have been summarized into the four rules of Lipinski (generally known as the “rules of fives” because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict bioavailability of compound during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present invention.

The four “rules of five” state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8 Å to about 15 Å. It will be understood that a candidate molecule, or even a selected molecule, may not meet all, or even any, of these characterizations. Nonetheless, the above guidelines are helpful in drug screening and design.

As explained above, the number of molecules identified as hits to the pharmacophore depend, at least in part, on the size of the database and the restrictiveness of the pharmacophore query. The number of molecules identified as hits from a pharmacophore query can be reduced by further modeling of fit to the binding site of the target biomolecule. Such modeling can be according to docking and scoring methods, as described below.

Docking and Scoring

Candidate molecules identified as being complementary to certain features of a target biomolecule as compared to a pharmacophore model (e.g., through a pharmacophore query as described above) can be further selected according to docking affinity for the target biomolecule (see e.g., Examples 6-7). In addition to pharmacophore model generation for database queries, a second sequential and complementary method for compound identification and design can be employed. Pharmacophore queries can filter out compounds quickly and docking and scoring can evaluate ligand-target biomolecule binding more accurately. In the case of protein or enzyme target biomolecules, amino acid residues of different domains in an inactive conformation can be used to define the docking site.

In various embodiments, selected compounds from the pharmacophore queries are docked to the target binding site using software designed for such analysis (e.g., Glide (Schrodinger, NY). Docking affinity can be calculated as numerical values (e.g., “Glide score”) based upon, for example, energy gained upon interaction of the molecule with the protein (e.g., “g_score”) and/or energy required to attain the docked conformation relative to the lowest energy conformation (e.g., “e_model”) (see e.g., Examples 6-7). For these particular examples, the more negative the score, the better the docking. Preferably, the g_score is less than about −5. Preferably, the e_model score is less than about −30. It is contemplated that the desirable numerical quantification of docking can vary between different target biomolecules.

In various embodiments, a threshold docking score (e.g., g_score and/or e_model score) can be chosen so as to manage the number of molecules for acquisition and further testing. For example, in some docking studies, a g-score of negative 5.0 (or greater magnitude in a negative direction) is considered a desirable docking score and the cut off is adjusted accordingly. As another example, in some docking studies, a g-score of negative 7.5 (or greater magnitude in a negative direction) is considered a desirable docking score and the cut off is adjusted accordingly. Thus, the magnitude of the g_score can be used to adjust a number of hits to a workable number that can be acquired and tested. As an example, if the total number of compounds identified from a pharmacophore query was about 1,000 to about 3,000, the docking scores can be used to rank such compounds so as to select about 100 to about 200 for further testing. It is contemplated the number of compounds to be selected for further testing could be lower or higher than these estimates. Preferably, magnitude of the g_score is used as a selection criteria, but it is contemplated that e_model score could be similarly used, especially where e_model score is of low magnitude. It is further contemplated that the selection criteria can be based upon both g_score and e_model score, preferably weighted toward g_score.

Docking and scoring can result in a group of compounds with multiple conformers. Using suitable modeling software (e.g., MOE), 3D structures can be converted to 2D and duplicates thereby removed. The resulting list of preferred chemical structures can used to search for commercial vendors using, for example, search engines designed for such a task (e.g., eMolecules.com).

Effect on Target Biomolecule

Candidate molecules selected according to pharmacophore query and/or further selected according to docking analysis can be tested for effect on the target biomolecule. Assessment of effect of a molecule on biomolecule function (e.g., inhibition of enzymatic activity) can be assessed by various methods known in the art (see e.g., Examples 1-3). For example, inhibitory effect of a candidate molecule on the catalytic activity of a target enzyme can be assessed by known activity assays specific for the target enzyme (see e.g., Reymond, ed. (2006) Enzyme Assays: High-throughput Screening, Genetic Selection and Fingerprinting, John Wiley & Sons, 386 p., ISBN-10: 3527310959; Eisenthall and Danson, Ed. (2002) Enzyme Assays, 2d edition, Oxford University Press, 384 p., ISBN-10: 0199638209). As described herein, an in-cell Western (ICW) screening protocol can be used to evaluate candidate compounds (see e.g., Example 1; Chen et al. (2005) Analytical Biochemistry 338, 136-142). Also as described herein, a MTT Cell Proliferation Assay can be used to evaluate candidate compounds (see e.g., Example 2). Also as described herein, an EGF inhibitor assay can be used to evaluate candidate compounds (see e.g., Example 3; Mukku (1984) J. Biol. Chem. 259, 6543-6546; Duh et al. (1990) World J. Surgery 14, 410-418; Lokeshwar et al. (1989) J. Biol. Chem. 264(32), 19318-19326).

Further Refinement

Further refinement of candidate molecules can be conducted. For example, data from biological assays can be correlated with the docking model so as to further refine lead-like molecules and/or drug-like molecules. Various software packages (e.g., MOE) can be employed to visualize active compound interaction with a target biomolecule to identify sites on the template suitable for modification by de novo design. Analogs of active compounds can be identified using similarity and sub-structure searches (see e.g., SciFinder; eModel). Available analogs can be analyzed according to docking and scoring procedures described above. Analogs with desirable docking scores can be acquired and further tested for biological effect on the target biomolecule according to methods described herein. One skilled in the art will understand these, and other, methods of refining and further developing candidate molecules identified by the methods presented herein.

Pharmacophores

Provided herein are a series of pharmacophores that can be used to identify small molecules that can substantially maintain a non-extended tether inactive configuration of EGFR or substantially prevent stabilization of an extended tether active configuration of EGFR. Pharmacophores include, but are not limited to, a Scheme II pharmacophore (AD4-1734-like), a Scheme III pharmacophore (AD4-1886-like), a Scheme IV pharmacophore (AD4-10381-like), and Scheme V pharmacophore (AD4-11091-like).

Scheme II Pharmacophore (AD4-1734-Like)

A Scheme II pharmacophore (AD4-1734-like) can include functional groups F(II)1, F(II)2, F(II)3, F(II)4, F(II)5, F(II)6, F(II)7, F(II)8, and F(II)9.

Functional group F(II)1 donates an H-bond or forms a salt bridge to a carboxylate side chain of receptor Asp553 of SEQ ID NO: 1 and has coordinates of r=56.363, θ (theta)=94.368, and Φ (phi)=−17.752 and a spherical radius of about 1.2 Å,

Functional group F(II)2 is a donor and has coordinates of r=53.290, θ (theta)=101.494, and Φ (phi)=−23.244 and a spherical radius of about 1.0 Å.

Functional group F(II)3 forms a hydrophobic contact with a side chain of receptor Val568, imidazolidine side chain of receptor Pro552 and with a side chain of Met253 of SEQ ID NO: 1 and has coordinates of r=53.726, θ (theta)=97.830, and Φ (phi)=−18.377 and a spherical radius of about 1.7 Å.

Functional group F(II)4 forms a hydrophobic contact with side chain of receptor Val575, Met253, and with an imidazolidine ring of receptor Pro552 of SEQ ID NO: 1 and has coordinates of r=53.647, θ (theta)=103.844, and Φ (phi)=−20.990 and a spherical radius of about 1.4 Å.

Functional group F(II)5 donates an H-bond to a side chain hydroxyl of Thr570 of SEQ ID NO: 1 and has coordinates of r=51.093, θ (theta)=104.261, and Φ (phi)=−25.552 and a spherical radius of about 1.2 Å.

Functional group F(II)6 is a donor having directionality of F4 with respect to a backbone carbonyl of receptor Thr570 of SEQ ID NO: 1 and has coordinates of r=52.340, θ (theta)=103.980, and Φ (phi)=−27.461 and a spherical radius of about 1.5 Å.

Functional group F(II)7 accepts an H-bond from a receptor backbone NH of Ala573 of SEQ ID NO: 1 and has coordinates of r=51.383, θ (theta)=106.455, and Φ (phi)=−24.319 and a spherical radius of about 1.2 Å.

Functional group F(II)8 is an acceptor having directionality of F7 with respect to the backbone NH of receptor Ala573 of SEQ ID NO: 1 and has coordinates of r=52.861, θ (theta)=107.691, and Φ (phi)=−25.448 and a spherical radius of about 1.5 Å.

Functional group F(II)9 donates an H-bond to a backbone carbonyl of Asp563 and forms a salt bridge to a side chain carboxylate of receptor Asp563 of SEQ ID NO: 1 and has coordinates of r=57.688, θ (theta)=99.198, and Φ (phi)=−21.588 and a spherical radius of about 1.2 Å.

A selected candidate compound can substantially align with at least one of functional groups F(II)1, F(II)2, F(II)3, F(II)4, F(II)5, F(II)6, F(II)7, F(II)8, and F(II)9. For example, a selected candidate compound can substantially align with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine of functional groups F(II)1, F(II)2, F(II)3, F(II)4, F(II)5, F(II)6, F(II)7, F(II)8, and F(II)9. Preferably, a selected candidate compound can substantially align with at least five of functional groups F(II)1, F(II)2, F(II)3, F(II)4, F(II)5, F(II)6, F(II)7, F(II)8, and F(II)9.

Scheme III Pharmacophore (AD4-1886-Like) A Scheme III pharmacophore (AD4-1886-like) can include functional groups F(III)1, F(III)2, F(III)3, F(III)4, F(III)5, F(III)6, F(III)7, F(III)8, and F(III)9.

Functional group F(III)1 donates a hydrogen bond to a side chain carbonyl of receptor Gln408 and accepts a hydrogen bond from the side chain NH₂ of receptor Gln384 of SEQ ID NO: 1 and has coordinates of r=81.552, θ (theta)=41.243, and Φ (phi)=45.369 and a spherical radius of about 1.2 Å.

Functional group F(III)2 accepts a hydrogen bond from a side chain OH of receptor Ser440 of SEQ ID NO: 1 and has coordinates of r=87.287, θ (theta)=40.739, and Φ (phi)=51.781 and a spherical radius of about 1.2 Å.

Functional group F(III)3 accepts a hydrogen bond from a side chain OH of receptor Ser440 of SEQ ID NO: 1 and has coordinates of r=86.320, θ (theta)=41.915, and Φ (phi)=52.323 and a spherical radius of about 1.5 Å.

Functional group F(III)4 forms a favorable coulombic interaction with an imidazole side chain of receptor His409 of SEQ ID NO: 1 and has coordinates of r=85.870, θ (theta)=38.463, and Φ (phi)=41.650 and a spherical radius of about 1.2 Å.

Functional group F(III)5 forms a favorable coulombic interaction with an imidazole side chain of receptor His409 of SEQ ID NO: 1 and has coordinates of r=82.241, θ (theta)=37.431, and Φ (phi)=44.151 and a spherical radius of about 1.5 Å.

Functional group F(III)6 accepts a hydrogen bond from, or forms a salt bridge to, NH³⁺ of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=88.009, θ (theta)=37.822, and Φ (phi)=54.903 and a spherical radius of about 1.2 Å.

Functional group F(III)7 accepts a hydrogen bond from, or forms a salt bridge to, NH³⁺ of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=86.513, θ (theta)=36.889, and Φ (phi)=54.484 and a spherical radius of about 1.5 Å.

Functional group F(III)8 donates a hydrogen bond to a side chain carbonyl of receptor Gln408 of SEQ ID NO: 1 and has coordinates of r=81.552, θ (theta)=41.243, and Φ (phi)=45.369 and a spherical radius of about 1.2 Å.

Functional group F(III)9 donates a hydrogen bond to a side chain carbonyl of receptor Gln408 of SEQ ID NO: 1 and has coordinates of r=79.652, θ (theta)=40.928, and Φ (phi)=44.528 and a spherical radius of about 1.6 Å.

A selected candidate compound can substantially align with at least one of functional groups F(III)1, F(III)2, F(III)3, F(III)4, F(III)5, F(III)6, F(III)7, F(III)8, and F(III)9. For example, a selected candidate compound can substantially align with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine of functional groups F(III)1, F(III)2, F(III)3, F(III)4, F(III)5, F(III)6, F(III)7, F(III)8, and F(III)9. Preferably, a selected candidate compound can substantially align with at least seven of functional groups F(III)1, F(III)2, F(III)3, F(III)4, F(III)5, F(III)6, F(III)7, F(III)8, and F(III)9.

Scheme IV Pharmacophore (AD4-10381-Like)

A Scheme IV pharmacophore (AD4-10381-like) can include functional groups F(IV)1, F(IV)2, F(IV)3, F(IV)4, F(IV)5, F(IV)6, F(IV)7, F(IV)8, F(IV)9, and F(IV)10.

Functional group F(IV)1 accepts a hydrogen bond from receptor side chain OH of Thr239 of SEQ ID NO: 1 and has coordinates of r=49.686, θ (theta)=113.993, and Φ (phi)=−17.014 and a spherical radius of about 1.2 Å.

Functional group F(IV)2 accepts a hydrogen bond from receptor side chain OH of Thr239 of SEQ ID NO: 1 and has coordinates of r=48.071, θ (theta)=115.388, and Φ (phi)=−16.211 and a spherical radius of about 1.6 Å.

Functional group F(IV)3 forms a hydrophobic contact with a side chain of receptor Met576 and imidazolidine ring of Pro242 of SEQ ID NO: 1 and has coordinates of r=50.781, θ (theta)=113.121, and Φ (phi)=−17.520 and a spherical radius of about 1.2 Å.

Functional group F(IV)4 forms a hydrophobic contact with a side chain of receptor Met576 and imidazolidine ring of Pro242 of SEQ ID NO: 1 and has coordinates of r=52.021, θ (theta)=114.264, and Φ (phi)=−15.878 and a spherical radius of about 1.5 Å.

Functional group F(IV)5 forms a hydrophobic contact with a side chain of receptor Met576 and imidazolidine ring of Pro242 of SEQ ID NO: 1 and has coordinates of r=50.322, θ (theta)=111.253, and Φ (phi)=−15.426 and a spherical radius of about 1.2 Å.

Functional group F(IV)6 forms a hydrophobic contact with a side chain of receptor Met576 and imidazolidine ring of Pro242 of SEQ ID NO: 1 and has coordinates of r=51.575, θ (theta)=112.433, and Φ (phi)=−13.827 and a spherical radius of about 1.5 Å.

Functional group F(IV)7 forms a hydrophobic contact with a side chain of receptor Leu243 and a side chain of Thr239 of SEQ ID NO: 1 and has coordinates of r=47.767, θ (theta)=112.521, and Φ (phi)=−10.196 and a spherical radius of about 1.2 Å

Functional group F(IV)8 donates a hydrogen bond to a backbone carbonyl of His280 of SEQ ID NO: 1 and has coordinates of r=45.184, θ (theta)=112.328, and Φ (phi)=−8.875 and a spherical radius of about 1.2 Å

Functional group F(IV)9 forms a hydrophobic contact with a side chain of receptor Met244 and Leu243 of SEQ ID NO: 1 and has coordinates of r=49.512, θ (theta)=108.007, and Φ (phi)=−8.504 and a spherical radius of about 1.2 Å.

Functional group F(IV)10 forms a hydrophobic contact with a side chain of receptor Met244 of SEQ ID NO: 1 and has coordinates of r=50.431, θ (theta)=104.985, and Φ (phi)=−7.420 and a spherical radius of about 1.8 Å.

A selected candidate compound can substantially align with at least one of functional groups F(IV)1, F(IV)2, F(IV)3, F(IV)4, F(IV)5, F(IV)6, F(IV)7, F(IV)8, F(IV)9, and F(IV)10. For example, a selected candidate compound can substantially align with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten of functional groups F(IV)1, F(IV)2, F(IV)3, F(IV)4, F(IV)5, F(IV)6, F(IV)7, F(IV)8, F(IV)9, and F(IV)10. Preferably, a selected candidate compound can substantially align with at least eight of functional groups F(IV)1, F(IV)2, F(IV)3, F(IV)4, F(IV)5, F(IV)6, F(IV)7, F(IV)8, F(IV)9, and F(IV)10.

Scheme V Pharmacophore (AD4-11091-Like)

A Scheme V pharmacophore (AD4-11091-like) can include functional groups F(V)1, F(V)2, F(V)3, F(V)4, F(V)5, F(V)6, F(V)7, F(V)8, F(V)9, F(V)10, and F(V)11.

Functional group F(V)1 accepts a hydrogen bond from a side chain amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=85.329, θ (theta)=34.962, and χ (phi)=53.394 and a spherical radius of about 1.2 Å.

Functional group F(V)2 accepts a hydrogen bond from a side chain amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=87.180, θ (theta)=35.618, and Φ (phi)=53.267 and a spherical radius of about 1.5 Å.

Functional group F(V)3 accepts a hydrogen bond from a side chain amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=86.349, θ (theta)=34.560, and Φ(phi)=55.424 and a spherical radius of about 1.5 Å.

Functional group F(V)4 donates a hydrogen bond to a side chain carbonyl of receptor Gln411 of SEQ ID NO: 1 and has coordinates of r=83.958, θ (theta)=35.296, and Φ (phi)=51.296 and a spherical radius of about 1.2 Å

Functional group F(V)5 donates a hydrogen bond to a side chain carbonyl of receptor Gln411 of SEQ ID NO: 1 and has coordinates of r=82.060, θ (theta)=34.984, and Φ (phi)=50.365 and a spherical radius of about 1.5 Å.

Functional group F(V)6 donates a hydrogen bond to a side chain nitrogen of receptor Gln411 of SEQ ID NO: 1 and has coordinates of r=83.884, θ (theta)=36.166, and Φ (phi)=49.227 and a spherical radius of about 1.2 Å.

Functional group F(V)7 donates a hydrogen bond to a side chain nitrogen of receptor Gln411 of SEQ ID NO: 1 and has coordinates of r=82.006, θ (theta)=35.523, and Φ (phi)=49.076 and a spherical radius of about 1.5 Å.

Functional group F(V)8 accepts a hydrogen bond to a side chain imidazole of receptor His409 of SEQ ID NO: 1 and has coordinates of r=85.100, θ (theta)=38.590, and Φ (phi)=43.143 and a spherical radius of about 1.2 Å.

Functional group F(V)9 forms a favorable π-π interaction with a phenyl ring of receptor Phe412 of SEQ ID NO: 1 and has coordinates of r=84.418, θ (theta)=37.489, and Φ (phi)=46.968 and a spherical radius of about 1.2 Å.

Functional group F(V)10 forms a favorable π-π interaction with a phenyl ring of receptor Phe412 of SEQ ID NO: 1 and has coordinates of r=82.486, θ (theta)=38.027, and Φ (phi)=47.246 and a spherical radius of about 1.5 Å. and

Functional group F(V)11 forms a favorable hydrophobic interaction with a side chain of receptors Val417 and Ile448 of SEQ ID NO: 1 and has coordinates of r=84.438, θ (theta)=40.753, and Φ (phi)=48.557 and a spherical radius of about 1.2 Å.

Functional group F11 forms a favorable hydrophobic interaction with a side chain of receptors Val417 and Ile448 of SEQ ID NO: 1 and has coordinates of r=84.438, θ (theta)=40.753, and Φ (phi)=48.557 and a spherical radius of about 1.2 Å.

A selected candidate compound can substantially align with at least one of functional groups F(V)1, F(V)2, F(V)3, F(V)4, F(V)5, F(V)6, F(V)7, F(V)8, F(V)9, F(V)10, and F(V)11. For example, a selected candidate compound can substantially align with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or at least eleven of functional groups F(V)1, F(V)2, F(V)3, F(V)4, F(V)5, F(V)6, F(V)7, F(V)8, F(V)9, F(V)10, and F(V)11. Preferably, a selected candidate compound can substantially align with at least nine of functional groups F(V)1, F(V)2, F(V)3, F(V)4, F(V)5, F(V)6, F(V)7, F(V)8, F(V)9, F(V)10, and F(V)11.

One aspect provides a method for identifying an epidermal growth factor receptor (EGFR) inhibitor comprising: providing a pharmacophore comprising any one of Schemes II, III, IV, or V as input to a 3-dimensional database; comparing a three dimensional structure of a candidate compound to the three dimensional structure of the pharmacophore; selecting a candidate compound with a three dimensional structure that substantially aligns with six or more functional groups of any one of Schemes II, III, IV, or V; wherein, similarity between the three-dimensional structure of the candidate compound and the three-dimensional structure of the pharmacophore is indicative of an ability of the candidate compound to inhibit EGFR by substantially maintaining a tethered inactive configuration of EGFR or substantially preventing stabilization of the untethered active configuration of EGFR.

In some embodiments, the method further comprises determining identity and spatial orientation of at least a portion of atoms of EGFR associated with stabilizing a tethered configuration of domain II and domain IV of EGFR in an inactive conformation; and constructing a pharmacophore, wherein the pharmacophore comprises a pluralitiy of pharmacophoric features that approximates the identity and the spatial orientation of the at least a portion of atoms of EGFR associated with stabilizing a tethered configuration of domain II and domain IV of EGFR in an inactive conformation such that the pharmacophore structural features are complementary to the inactive EGFR configuration.

In some embodiments, determining identity and spatial orientation of at least a portion of atoms of EGFR associated with stabilizing a tethered configuration of domain II and domain IV of EGFR in an inactive conformation comprises analysis of X-ray crystallographic data derived from a crystalline form of EGFR in an inactive, tethered conformation.

In some embodiments, at least one pharmacophoric feature approximates identity and spatial orientations of at least a portion of atoms of domain II of EGFR in a tethered inactive conformation. In some embodiments, at least one pharmacophoric feature approximates identity and spatial orientations of at least a portion of atoms of a cleft region between domain II and domain IV of EGFR in a tethered inactive conformation.

In some embodiments, the method further comprises determining a docking affinity of the candidate molecule for the at least a portion of atoms of EGFR associated with stabilizing a tethered configuration of domain II and domain IV of EGFR in an inactive conformation; wherein docking affinity is quantified by energy gained upon interaction of the candidate molecule with the target biomolecule, energy required to attain the docked conformation relative to the lowest energy conformation, or a combination thereof.

Compounds

Another aspect of the present invention includes small molecule compounds, identified by the methods described herein. Compounds described herein can have an anti-proliferative effect useful in, for example, treating a proliferative disease, disorder, or condition. Compounds described herein can be useful for the treatment of diseases, disorders, or conditions related to a target biomolecule according to which they were identified from. Various embodiments of compounds described herein can hold multiple domain proteins in a tethered, inactive state. For example, it is well known that inhibition of growth factor proteins has a benefit in treatment of certain conditions in oncology. As another example, inhibition of EGFR has a benefit in treatment of certain conditions associated with EGFR, as discussed further below. Compounds described herein can have an EGFR inhibitory effect useful in, for example, treating a proliferative disease or disorder associated with EGFR. Compounds described herein have been demonstrated to be empirically effective in treating proliferative diseases and conditions.

Various compounds, including AD4-1734, AD4-1886, AD4-10381, and AD4-10381, were identified as EGFR inhibitors through the pharmacophoric approach described herein (see e.g., Examples 4-5). Such compounds, and derivatives thereof, have utility as therapeutic agents for treatment of proliferative diseases or conditions. For example, compounds described herein can be used as a therapeutic agent for the treatment of an EGFR-associated disease, disorder, or condition. Analogs and derivatives of such compounds are expected to have the same or similar anti-proliferative effects and utility (see e.g., Examples 6-7). Identified compounds and analogs and derivatives thereof are further discussed below.

While under no obligation to provide an underlying mechanism and in no way limiting the present invention by doing so, it is presently thought that at least a portion of activity of compounds described herein arise from inhibition of EGFR. It is further contemplated that the presently described compounds may have additional modes of action in their effectiveness in treating a proliferative disease, disorder, or condition. Regardless of the underlying mechanism, compounds described herein have been demonstrated to be empirically effective in treating proliferative diseases and conditions.

The following definitions are provided to better define the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

The expression “alkyl”, unless specifically limited, denotes a C₁₋₁₂ alkyl group, suitably a C₁₋₆ alkyl group, e.g. C₁₋₄ alkyl group. Alkyl groups may be straight chain or branched. Suitable alkyl groups include, for example, methyl, ethyl, propyl (e.g. n-propyl and isopropyl), butyl (e.g. n-butyl, iso-butyl, sec-butyl and tert-butyl), pentyl (e.g. n-pentyl), hexyl (e.g. n-hexyl), heptyl (e.g. n-heptyl) and octyl (e.g. n-octyl). The expression “alk”, for example in the expressions “alkoxy”, “haloalkyl” and “thioalkyl” should be interpreted in accordance with the definition of “alkyl”. Exemplary alkoxy groups include methoxy, ethoxy, propoxy (e.g. n-propoxy), butoxy (e.g. n-butoxy), pentoxy (e.g. n-pentoxy), hexoxy (e.g. n-hexoxy), heptoxy (e.g. n-heptoxy) and octoxy (e.g. n-octoxy).

The expression “cycloalkyl”, unless specifically limited, denotes a C₃₋₁₀ cycloalkyl group (i.e., 3 to 10 ring carbon atoms), more suitably a C₃₋₈ cycloalkyl group, for example, a C₃₋₆ cycloalkyl group. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. A preferred number of ring carbon atoms is three to six.

The expression “aryl”, unless specifically limited, denotes a C₆₋₁₂ aryl group, suitably a C₆₋₁₀ aryl group, more suitably a C₆₋₈ aryl group. Aryl groups will contain at least one aromatic ring (e.g. one, two or three rings). An example of a typical aryl group with one aromatic ring is phenyl. An example of a typical aryl group with two aromatic rings is naphthyl.

The expression “heteroaryl”, unless specifically limited, denotes an aryl residue, wherein one or more (e.g., 1, 2, 3, or 4, suitably 1, 2 or 3) ring atoms are replaced by heteroatoms selected from N, S and O, or else a 5-membered aromatic ring containing one or more (e.g., 1, 2, 3, or 4, suitably 1, 2 or 3) ring atoms selected from N, S and O. Exemplary monocyclic heteroaryl groups having one heteroatom include: five membered rings (e.g., pyrrole, furan, thiophene); and six membered rings (e.g., pyridine, such as pyridin-2-yl, pyridin-3-yl and pyridin-4-yl). Exemplary monocyclic heteroaryl groups having two heteroatoms include: five membered rings (e.g., pyrazole, oxazole, isoxazole, thiazole, isothiazole, imidazole, such as imidazol-1-yl, imidazol-2-yl imidazol-4-yl); six membered rings (e.g., pyridazine, pyrimidine, pyrazine). Exemplary monocyclic heteroaryl groups having three heteroatoms include: 1,2,3-triazole and 1,2,4-triazole. Exemplary monocyclic heteroaryl groups having four heteroatoms include tetrazole. Exemplary bicyclic heteroaryl groups include: indole (e.g., indol-6-yl), benzofuran, benzothiophene, quinoline, isoquinoline, indazole, benzimidazole, benzthiazole, quinazoline and purine.

A saturated group is generally understood as having no double or triple bonds. For example, in a saturated linear hydrocarbon, each carbon atom is attached to two hydrogen atoms, except those at the ends of the chain, which bear three hydrogen atoms. For example, an unsaturated hydrocarbon is generally understood as a carbon structure containing one or more double or triple bonds.

The term “halogen” or “halo” includes fluorine (F), chlorine (Cl) bromine (Br) or iodine (I).

The term “amino” refers to the group —NH₂.

All possible stereoisomers of the claimed compounds are included in the present disclosure. Where a compound described herein has at least one chiral center, it may accordingly exist as enantiomers. Where a compound possess two or more chiral centers it may additionally exist as diastereomers. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present disclosure.

In view of the close relationship between the free compounds and the compounds in the form of their salts, whenever a compound is referred to in this context, a corresponding salt is also intended, provided such is possible or appropriate under the circumstances. The pharmaceutically acceptable salt can take a form in which a basic side chain is protonated with an inorganic or organic acid. Representative organic or inorganic acids include hydrochloric, hydrobromic, perchloric, sulfuric, nitric, phosphoric, acetic, propionic, glycolic, lactic, succinic, maleic, fumaric, malic, tartaric, citric, benzoic, mandelic, methanesulfonic, hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, salicylic, saccharinic or trifluoroacetic acid. Alternatively it may take the form in which an acidic side chain forms a salt with a metal ion (e.g., sodium, potassium ions and the like) or other positive ion such as ammonium. All pharmaceutically acceptable acid addition salt forms of the compounds described herein are intended to be embraced by the scope of this disclosure.

Some of the crystalline forms of the compounds may exist in more than one polymorphic form and as such all forms are intended to be included in the present disclosure. In addition, some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are also intended to be encompassed within the scope of this disclosure. The compounds, including their salts, can also be obtained in the form of their hydrates, or include other solvents used for their crystallization.

The present disclosure further includes within its scope prodrugs of the compounds described herein. In general, such prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the desired therapeutically active compound. Thus, in these cases, the methods of treatment of the present invention, the term “administering” shall encompass the treatment of the various disorders described with prodrug versions of one or more of the claimed compounds, but which converts to the above specified compound in vivo after administration to the subject.

As used herein, the term “composition” is intended to encompass a product comprising a claimed compound(s) in a therapeutically effective amount, as well as any product which results, directly or indirectly, from combinations of the claimed compounds.

AD4-1734

AD4-1734 is identified as an inhibitor of epidermal growth factor binding to its receptor (see e.g., Example 5).

As described herein, a pharmacophore model was utilized to identify small molecules that are AD4-1734-like.

Type P AD4-1734-Like

One structure derived from the AD4-1734-like pharmacophore is “Type P” as follows:

In the above structure, R²³ and R²⁷ of Formula (13) can be hydrogen.

R²⁴ and R²⁶ of Formula (13) can be independently selected from represent hydrogen, lower alkyl (one to six carbon, straight chain, branched, or optionally containing unsaturation), alkoxy (—OR¹⁰ where R¹⁰ is defined as a lower alkyl group or cycloalkyl group in the above definition), or halogen (F, Cl, Br, or I). Preferably, R²⁴ and R²⁶ of Formula (13) are independently selected from hydrogen or alkoxy.

R²⁵ of Formula (13) can represent hydrogen, lower alkyl, cycloalkyl, substituted alkyl, alkoxy or halogen. Preferably, R²⁵ of Formula (13) is selected from hydrogen or alkoxy. Substituted alkyl is defined as a hydrocarbon chain with 1-3 carbons which is substituted with additional functional groups from the following list: carboxyl defined as —COOR¹⁰, (R¹⁰ is a lower alkyl group or cycloalkyl group as in the above definition), aryl, aryloxy (—OAr). Aryl is defined as an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more of the following groups: lower alkyl defined as C-1 to C-4, straight chain, branched, or optionally containing unsaturation; cycloalkyl, which is defined as C-1 to C-6 optionally containing unsaturation; Aryl including phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; Alkoxy (—OR¹⁰ where R¹⁰ is defined as a lower alkyl group or cycloalkyl group in the above definition); Trifluoromethyl, Trifluoromethoxy, Difluoromethoxy, 3,4-methylenedioxy, 2,3-methylenedioxy, Nitro or Halogen (F, Cl, Br, I).

R²⁸ of Formula (13) can be selected from hydrogen, lower alkyl or aryl. Preferably, R²⁸ of Formula (13) is selected from hydrogen or aryl. More preferably, R²⁸ of Formula (13) is hydrogen. Aryl represents an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more of the following groups: lower alkyl defined as above, cycloalkyl defined as above, or halogen.

R²⁹ of Formula (13) can be selected from hydrogen, lower alkyl, hydroxy, alkoxy or halogen. Lower alkyl, alkoxy, and halogen are as defined above.

R³⁰ of Formula (13) can be selected from hydrogen, lower alkyl, alkoxy, trifluoromethyl, or halogen. Preferably, R³⁰ of Formula (13) is lower alkyl. Lower alkyl, alkoxy, and halogen are as defined above.

R³¹ of Formula (13) can be selected from hydrogen, hydroxy, alkoxy, lower alkyl, trifluoromethyl or halogen. Preferably, R³¹ of Formula (13) is selected from hydroxy or alkoxy. Lower alkyl, alkoxy, and halogen are as defined above.

R³² of Formula (13) can be selected from hydrogen, lower alkyl, hydroxy, trifluoromethyl or halogen. Preferably, R³² of Formula (13) is hydrogen. Lower alkyl and halogen are as defined above.

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (13).

In some embodiments, the compound of Formula (13) is AD4-10631.

In some embodiments, the compound of Formula (13) is AD4-10174.

In some embodiments, the compound(s) of Formula (13) excludes compound AD4-1734, Formula (12).

Type Q AD4-1734-Like

One structure derived from the AD4-1734-like pharmacophore is “Type Q” as follows:

In the above structure, R³³ of Formula (14) can be selected from lower alkyl, substituted lower alkyl, or aryl. Preferably R³³ of Formula (14) is aryl. Lower alkyl, substituted lower alkyl, and aryl are as defined above.

R³⁴ of Formula (14) can be selected from hydrogen.

R³⁵ of Formula (14) can be selected from hydrogen, hydroxy, alkoxy, lower alkyl, trifluoromethyl or halogen. Preferably R³⁵ of Formula (14) is lower alkyl. Lower alkyl, alkoxy, and halogen are as defined above.

R³⁶ of Formula (14) can be selected from hydrogen, lower alkyl, hydroxy, alkoxy, trifluoromethyl or halogen. Preferably, R³⁶ of Formula (14) is selected from hydroxy or alkoxy. Lower alkyl and halogen are as defined above.

R³⁷ of Formula (14) can be selected from hydrogen.

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (14).

In some embodiments, the compound of Formula (14) is AD4-10188.

In some embodiments, the compound(s) of Formula (14) excludes compound AD4-1734, Formula (12).

Type R AD4-1734-Like

One structure derived from the AD4-1734-like pharmacophore is “Type R” as follows:

In the above structure, X² and X³ of Formula (15) can be independently selected from carbon, oxygen, nitrogen or sulfur atom. Preferably, X² and X³ of Formula (15) are independently selected from oxygen or carbon. In Formula (15), n can be one or two resulting in a 5 or 6 membered ring. Preferably, n of Formula (15) is n=1.

R³⁸ of Formula (15) can be selected from hydrogen.

R³⁹ of Formula (15) can be as defined for R²⁴.

R⁴⁰ of Formula (15) can be as defined for R²³.

R⁴¹ of Formula (15) can be selected from hydrogen, lower alkyl or aryl.

R⁴² of Formula (15) can be selected from hydrogen, lower alkyl, hydroxy, alkoxy, or halogen. Preferably, R⁴² of Formula (15) is hydrogen. Lower alkyl, alkoxy and halogen are as defined above.

R⁴³ of Formula (15) can be selected from hydrogen, lower alkyl, alkoxy, trifluoromethyl, or halogen. Preferably R⁴³ of Formula (15) is lower alkyl. Lower alkyl, alkoxy and halogen are as defined above.

R⁴⁴ of Formula (15) can be selected from hydrogen, hydroxy, alkoxy, lower alkyl, trifluoromethyl or halogen. Preferably, R⁴⁴ of Formula (15) is selected from hydroxy or alkoxy. Lower alkyl, alkoxy and halogen are as defined above.

R⁴⁵ of Formula (15) can be selected from hydrogen, lower alkyl, hydroxy, trifluoromethyl, or halogen. Preferably, R⁴⁵ of Formula (15) is hydrogen. Lower alkyl and halogen are as defined above.

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (15).

In some embodiments, the compound of Formula (15) is AD4-10633.

In some embodiments, the compound(s) of Formula (15) excludes compound AD4-1734, Formula (12).

Type S AD4-1734-Like

One structure derived from the AD4-1734-like pharmacophore is “Type S” as follows:

In the above structure, X⁴ and X⁵ of Formula (16) can be independently selected from carbon, oxygen, nitrogen or sulfur atom. Preferably, X⁴ and X⁵ of Formula (16) are independently selected from oxygen or carbon. In Formula (16), n can be one or two resulting in a 5 or 6 membered ring. Preferably, n of Formula (16) is n=1.

R⁴⁶ and R⁵⁰ of Formula (16) can be independently selected from hydrogen.

R⁴⁷ and R⁴⁹ of Formula (16) can be independently selected from hydrogen, lower alkyl, alkoxy or halogen. Lower alkyl, alkoxy and halogen are as defined above.

R⁴⁸ of Formula (16) can be selected from hydrogen, lower alkyl, cycloalkyl, substituted alkyl, alkoxy, or halogen. Preferably, R⁴⁸ of Formula (16) is selected from hydrogen or lower alkyl. Lower alkyl, cycloalkyl, substituted alkyl, and alkoxy are as defined above. Halogen is F, Cl, Br, or I.

Alternatively, R⁴⁷ and R⁴⁸ of Formula (16) can be joined to form a ring as described for Formula (15). Preferably, R⁴⁷ and R⁴⁸ of Formula (16) are joined to form a ring a 5-membered ring.

R⁵¹ of Formula (16) can be selected from hydrogen, lower alkyl, or aryl. Preferably, R⁵¹ of Formula (16) is selected from hydrogen or aryl. Lower alkyl or aryl are as defined above.

R⁵² of Formula (16) can be selected from hydrogen, lower alkyl, hydroxy, alkoxy or halogen. Preferably, R⁵² of Formula (16) is hydrogen. Lower alkyl, alkoxy and halogen are as defined above.

R⁵³ of Formula (16) can be selected from hydrogen, lower alkyl, hydroxy, trifluoromethyl or halogen. Preferably, R⁵³ of Formula (16) is hydrogen. Lower alkyl and halogen are as defined above.

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (16).

In some embodiments, the compound of Formula (16) is AD4-10639.

In some embodiments, the compound of Formula (16) is AD4-10179.

In some embodiments, the compound(s) of Formula (16) excludes compound AD4-1734, Formula (12).

AD4-1886

AD4-1886 is identified as an inhibitor of epidermal growth factor binding to its receptor (see e.g., Example 4).

As described herein, a pharmacophore model was utilized to identify small molecules that are AD4-1886-like.

Type J AD4-1886-Like

One structure derived from the AD4-1886-like pharmacophore is “Type J” as follows:

In the above structure, R⁵⁴ of Formula (18) is a 5 or 6 membered heterocyclic containing from 1 to 4 N, O, or S atoms or any combination of those atoms with carbon atoms to form a heterocyclic aromatic ring which is optionally substituted with from 1 to 3 of the following groups: lower alkyl, cycloalkyl, alkoxy (—OR¹⁰ where R¹⁰ is defined as a lower alkyl group or cycloalkyl group in the above definition), trifluoromethyl, trifluoromethoxy, substituted amino, nitro or halogen (F, Cl, Br, or I). Lower Alkyl is defined as one to six carbon, straight chain, branched, or optionally containing unsaturation. Cycloalkyl is defined as C-1 to C-6 optionally containing unsaturation. As preferred examples, R⁵⁴ of Formula (18) can represent: 4,6-Dimethyl-2-pyrimidine, 2,6-Dimethoxy-4-pyrimidine, 6-Methoxy-4-pyrimidine, 5-Ethyl-2-(1,3,4-Thiadiazole), 5-Methyl-3-Isoxazole, 3-Methoxy-6-pyridazinamine, 2-Thiazole or 2-Methoxy-3-pyrazine.

R⁵⁵ of Formula (18) can be selected from hydrogen or lower alkyl. Preferably, R⁵⁵ of Formula (18) is selected from hydrogen or methyl. Lower alkyl is as defined above.

R⁵⁶, R⁵⁷, R⁵⁸, and R⁵⁹ of Formula (18) can be independently selected from hydrogen.

R⁶⁰ of Formula (18) can be selected from lower alkyl, cycloalkyl, or substituted alkyl. Lower alkyl and cycloalkyl are as defined above. Substituted alkyl is defined as a hydrocarbon chain with 1-3 carbons which is substituted with additional functional groups from the following list: carboxyl defined as —COOR¹⁰ (R¹⁰ is a lower alkyl group or cycloalkyl group as in the above definition), aryl, aryloxy (—OAr) where aryl is defined as an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more of the following groups: lower alkyl defined as C-1 to C-4, straight chain, branched, or optionally containing unsaturation, cycloalkyl defined as C-1 to C-6 optionally containing unsaturation, aryl including phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms, alkoxy (—OR¹⁰ where R¹⁰ is defined as a lower alkyl group or cycloalkyl group in the above definition), Trifluoromethyl, Trifluoromethoxy, Difluoromethoxy, 3,4-methylenedioxy, 2,3-methylenedioxy, Nitro or Halogen (F, Cl, Br, I).

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (18).

In some embodiments, the compound of Formula (18) is AD4-12158.

In some embodiments, the compound of Formula (18) is AD4-12267.

In some embodiments, the compound of Formula (18) is AD4-11384.

In some embodiments, the compound(s) of Formula (18) excludes compound AD4-1886, Formula (17).

Type K AD4-1886-Like

One structure derived from the AD4-1886-like pharmacophore is “Type K” as follows:

In the above structure, R⁶¹, R⁶², and R⁶⁴ of Formula (19) can be independently selected from hydrogen or lower alkyl. Lower alkyl is as defined above.

R⁶³ and R⁶⁵ of Formula (19) can be selected from hydrogen, lower alkyl or alkoxy. Lower alkyl and alkoxy are as defined above.

R⁶⁶ of Formula (19) can be selected from hydrogen or lower alkyl. Lower alkyl is as defined above.

R⁶⁷, R⁶⁸, R⁶⁹, and R⁷⁰ of Formula (19) can be independently selected from hydrogen.

R⁷¹ of Formula (19) can be selected from lower alkyl, cycloalkyl, aryl, substituted alkyl, or heteraryl. Lower alkyl, cycloalkyl, aryl, and substituted alkyl are as defined above. Hereoaryl is defined as a heterocyclic ring containing from 1 to 4 N, O, or S atoms which has optional substitution with one or more of the following functional groups: lower alkyl defined as C-1 to C-4, straight chain, branched, or optionally containing unsaturation, cycloalkyl defined as C-1 to C-6 optionally containing unsaturation, aryl including phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms, alkoxy (—OR¹⁰ where R¹⁰ is defined as a lower alkyl group or cycloalkyl group in the above definition), trifluoromethyl, trifluoromethoxy, nitro or halogen (F, Cl, Br, I).

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (19).

In some embodiments, the compound of Formula (19) is AD4-11409.

In some embodiments, the compound(s) of Formula (19) excludes compound AD4-1886, Formula (17).

Type L AD4-1886-Like

One structure derived from the AD4-1886-like pharmacophore is “Type L” as follows:

In the above structure, R⁷² of Formula (20) can be selected from Hydrogen or Lower Alkyl. Lower alkyl is as defined above.

R⁷³ of Formula (20) can be selected from lower alkyl, cycloalkyl, substituted alkyl, or acyl. Lower alkyl, cycloalkyl, and substituted alkyl are as defined above. Acyl represents a substituted ketone —COR¹⁰ where R¹⁰ is defined as a lower alkyl group or cycloalkyl group in the above definition.

R⁷⁴, R⁷⁵, R⁷⁶, and R⁷⁷ of Formula (20) can be independently selected from hydrogen.

R⁷⁸ of Formula (20) can be selected from lower alkyl, cycloalkyl, and substituted alkyl. Lower alkyl, cycloalkyl, and substituted alkyl are as defined above.

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (20).

In some embodiments, the compound of Formula (20) is AD4-10608.

In some embodiments, the compound of Formula (20) is AD4-10609.

In some embodiments, the compound(s) of Formula (20) excludes compound AD4-1886, Formula (17).

Type M AD4-1886-Like

One structure derived from the AD4-1886-like pharmacophore is “Type M” as follows:

In the above structure, n of Formula (21) can be 1 or 2, which represents one or two methylene groups resulting in either a five or six membered fused ring system.

R⁷⁹, R⁸¹, R⁸², R⁸³, R⁸⁴, R⁸⁵, and R⁸⁶ of Formula (21) can be independently selected from hydrogen.

R⁸⁰ of Formula (21) can be selected from hydrogen, lower alkyl, alkoxy, halogen, or trifluoromethyl. Lower alkyl and alkoxy are as defined above. Halogen is defined as F, Cl, Br, or I.

R⁸⁷ of Formula (21) can be selected from lower alkyl, cycloalkyl, substitute alkyl, or aryl. Lower alkyl, cycloalkyl, substitute alkyl, and aryl are as defined above.

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (21).

In some embodiments, the compound of Formula (21) is AD4-10626.

In some embodiments, the compound(s) of Formula (21) excludes compound AD4-1886, Formula (17).

Type N AD4-1886-Like

One structure derived from the AD4-1886-like pharmacophore is “Type N” as follows:

In the above structure, n of Formula (22) can be 3, 4 or 5, which represents 3, 4, or 5 methylene groups resulting in either a five, six, or seven membered ring (pyrrolidine, piperidine, or hexahydro-1H-azepine).

R⁸⁸ of Formula (22) can be selected from hydrogen or lower alkyl. Lower alkyl is as defined above.

R⁸⁹, R⁹⁰, R⁹¹, R⁸³, and R⁹² of Formula (22) can be independently selected from hydrogen.

R⁹³ of Formula (22) can be selected from lower alkyl, cycloalkyl, substituted alkyl, or aryl. Lower alkyl, cycloalkyl, substituted alkyl, and aryl are as defined above.

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (22).

In some embodiments, the compound of Formula (22) is AD4-11970.

In some embodiments, the compound of Formula (22) is AD4-12107.

In some embodiments, the compound(s) of Formula (22) excludes compound AD4-1886, Formula (17).

Type O AD4-1886-Like

One structure derived from the AD4-1886-like pharmacophore is “Type O” as follows:

In the above structure, R⁹⁴ of Formula (23) is a 5 or 6 membered heterocyclic containing from 1 to 4 N, O, or S atoms or any combination of those atoms with carbon atoms to form a heterocyclic aromatic ring which is optionally substituted with from 1 to 3 of the following groups: lower alkyl, cycloalkyl, alkoxy (—OR¹⁰ where R¹⁰ is defined as a lower alkyl group or cycloalkyl group in the above definition), trifluoromethyl, trifluoromethoxy, substituted amino, nitro or halogen (F, Cl, Br, or I). Lower alkyl is defined as one to six carbon, straight chain, branched, or optionally containing unsaturation. Cycloalkyl is defined as C-1 to C-6 optionally containing unsaturation.

R⁹⁵ of Formula (23) can be selected from hydrogen or lower alkyl. Lower alkyl is as defined above.

R⁹⁶, R⁹⁷, R⁹⁸, and R⁹⁹ of Formula (23) can be independently selected from hydrogen.

R¹⁰⁰ of Formula (23) can be selected from aryl or heteroaryl. Aryl and heteroaryl are as defined above.

In some embodiments, the compound of Formula (23) is AD4-11883.

In some embodiments, the compound of Formula (23) is AD4-11638.

In some embodiments, the compound of Formula (23) is AD4-11645.

In some embodiments, the compound(s) of Formula (23) excludes compound AD4-1886, Formula (17).

AD4-10381

AD4-10381 is identified as an inhibitor of epidermal growth factor binding to its receptor (see e.g., Example 5).

As described herein, a pharmacophore model was utilized to identify small molecules that are AD4-10381-like.

Type D AD4-10381-Like

One structure derived from the AD4-10381-like pharmacophore is “Type D” as follows:

In the above structure, R¹⁰¹ of Formula (25) can be selected from lower alkyl, cycloalkyl or aryl. Preferably, R¹⁰¹ of Formula (25) is selected from methyl or phenyl. Lower alkyl is defined as one to six carbon, straight chain, branched, or optionally containing unsaturation. Cycloalkyl is defined as three to six membered carbocyclic ring optionally containing unsaturation. Aryl represents an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more of the following groups: lower alkyl defined as above, cycloalkyl defined as above, or heteroaryl containing from 1 to 4 N, O, or S atoms.

R¹⁰² of Formula (25) can be selected from hydrogen.

R¹⁰³ of Formula (25) can be selected from hydrogen, lower alkyl, cycloalkyl, alkoxy, or halogen. Preferably, R¹⁰³ of Formula (25) is selected from methyl, ethyl, chloro, methoxy, or ethoxy. Lower alkyl is defined as above. Cycloalkyl is defined as above. Alkoxy is defined as —OR¹⁰ where R¹⁰ is defined as a lower alkyl group or cycloalkyl group in the above definition.

R¹⁰⁴ of Formula (25) can be selected from hydrogen, lower alkyl, cycloalkyl, or alkoxy. Preferably, R¹⁰⁴ of Formula (25) is selected from hydrogen, methyl, or methoxy. Lower alkyl is defined as above. Cycloalkyl is defined as above. Alkoxy is defined as above.

R¹⁰⁵ of Formula (25) can be selected from hydrogen, lower alkyl, cycloalkyl, or alkoxy. Preferably, R¹⁰⁵ of Formula (25) is selected from hydrogen, methyl, methoxy, or ethoxy. Lower alkyl is defined as above. Cycloalkyl is defined as above. Alkoxy is defined as above.

R¹⁰⁶ of Formula (25) can be selected from hydrogen, lower alkyl, aryl, or substituted alkyl. Preferably, R¹⁰⁶ of Formula (25) is selected from methyl or phenyl. Lower alkyl is defined as above. Substituted alkyl is defined as lower alkyl group with substitution of additional groups such as an aryl thioether, —S—Ar′, where Ar′ is defined as phenyl, substituted phenyl or an unsubstituted heteroaryl five or six membered ring containing from 1 to 4 N, O, or S atoms, or a heteroaryl five or six membered ring containing from 1 to 4 N, O, or S atoms which has one or more optional substitution with the substituent defined as one or more of the following groups: lower alkyl defined as above, cycloalkyl defined as above, unsaturation, bicyclic heterocycles such as benzimidazoles, benzoxazoles, benzthiazoles, or benzopyrazoles. Aryl is defined as an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more of the following groups: lower alkyl defined as C-1 to C-4, straight chain, branched, or optionally containing unsaturation, Cycloalkyl defined as C-1 to C-6 optionally containing unsaturation, aryl including phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms, Alkoxy (—OR¹⁰ where R¹⁰ is defined as a lower alkyl group or cycloalkyl group in the above definition), trifluoromethyl, trifluoromethoxy, difluoromethoxy, 3,4-methylenedioxy, 2,3-methylenedioxy, nitro or halogen (F, Cl, Br, I).

R¹⁰⁷ of Formula (25) can be selected from hydrogen, lower alkyl, cycloalkyl, aryl, or substituted alkyl. Preferably, R¹⁰⁷ of Formula (25) is methyl. Lower alkyl is defined as above. Cycloalkyl is defined as above. Aryl is defined as above. Substituted alkyl is defined as above.

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (25).

In some embodiments, the compound(s) of Formula (25) excludes compound AD4-10381, Formula (24).

In some embodiments, the compound of Formula (25) is AD4-11511.

In some embodiments, the compound of Formula (25) is AD4-12632.

In some embodiments, the compound of Formula (25) is AD4-10381.

In some embodiments, the compound of Formula (25) excludes AD4-10381.

Type E AD4-10381-Like

One structure derived from the AD4-10381-like pharmacophore is “Type E” as follows:

In the above structure, R¹⁰⁸ of Formula (27) can be selected from lower alkyl, substituted alkyl, cycloalkyl, aryl, amino, or hydroxyl. Preferably, R¹⁰⁸ of Formula (27) is selected from methyl, phenyl, or hydroxyl. Lower alkyl is defined as above. Substituted alkyl is defined as above. Cycloalkyl is defined as above. Aryl is defined as above.

R¹⁰⁹ of Formula (27) can be selected from hydrogen.

R¹¹⁰ of Formula (27) can be selected from hydrogen, lower alkyl, substituted alkyl, cycloalkyl, alkoxy, or halogen. Preferably, R¹¹⁰ of Formula (27) is selected from hydrogen, methyl, methoxy, or chloro. Lower alkyl is defined as above. Substituted alkyl is defined as above. Alkoxy is defined as above. Halogen is defined as F, Cl, Br, or I.

R¹¹¹ of Formula (27) can be selected from hydrogen, lower alkyl, substituted alkyl, cycloalkyl, alkoxy, or halogen. Preferably, R¹¹¹ of Formula (27) is selected from hydrogen, methyl, methoxy, or chloro. Lower alkyl is defined as above. Substituted alkyl is defined as above. Cycloalkyl is defined as above. Alkoxy is defined as above. Halogen is defined as above.

R¹¹² of Formula (27) can be selected from hydrogen or lower alkyl. Preferably, R¹¹² of Formula (27) is methyl. Lower alkyl is defined as above.

R¹¹³ of Formula (27) can be selected from lower alkyl, trifluoromethyl, aryl, or amino. Preferably, R¹¹³ of Formula (27) is selected from methyl, phenyl, or amino. Lower alkyl is defined as above. Aryl is defined as above.

R¹¹⁴ of Formula (27) can be selected from hydrogen, acyl or carboxyalkyl. Acyl represents a substituted ketone —COR¹⁰ where R¹⁰ is defined as a lower alkyl group or cycloalkyl group in the above definition. Carboxyalkyl is defined as a substituted ester group COOR¹⁰ where R¹⁰ is defined as a lower alkyl group or cycloalkyl group in the above definition.

R¹¹⁵ of Formula (27) can be selected from lower alkyl, trifluoromethyl, or aryl. Preferably, R¹¹⁵ of Formula (27) is methyl, phenyl or trifluoromethyl. Lower alkyl is defined as above. Aryl is defined as above.

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (27).

In some embodiments, the compound of Formula (27) is AD4-12681.

In some embodiments, the compound of Formula (27) is AD4-12679.

In some embodiments, the compound(s) of Formula (27) excludes compound AD4-10381, Formula (24).

Type F AD4-10381-Like

One structure derived from the AD4-10381-like pharmacophore is “Type F” as follows:

In the above structure, n of Formula (28) can be 1 or 2, which represents one or two methylene groups resulting in either a five or six membered carbocyclic ring. The six membered ring contains optional unsaturation resulting in a fused benzo-ring system as shown below (see Formula (29)).

R¹¹⁶ of Formula (28) or Formula (29) can be selected from lower alkyl or aryl. Preferably, R¹¹⁶ of Formula (28) or Formula (29) are independently methyl or phenyl. Lower alkyl is defined as above. Aryl is defined as above.

R¹¹⁷ of Formula (28) or Formula (29) can be selected from hydrogen.

R¹¹⁸ of Formula (28) or Formula (29) can be selected from hydrogen, lower alkyl, alkoxy, or halogen. Lower alkyl is defined as above. Alkoxy is defined as above. Halogen is defined as above.

R¹¹⁹ of Formula (28) or Formula (29) can be selected from hydrogen, lower alkyl, or aryl. Lower alkyl is defined as above. Aryl is defined as above.

R¹²⁰ of Formula (28) or Formula (29) can be selected from hydrogen, lower alkyl, or alkoxy. Lower alkyl is defined as above. Alkoxy is defined as above.

R¹²¹ of Formula (28) or Formula (29) can be selected from hydrogen or aryl. Aryl is defined as above.

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (28) or Formula (29).

In some embodiments, the compound of Formula (28) is AD4-11436.

In some embodiments, the compound(s) of Formula (28) and Formula (29) excludes compound AD4-10381, Formula (24).

AD4-11091

AD4-11091 is identified as an inhibitor of epidermal growth factor binding to its receptor (see e.g., Example 4).

As described herein, a pharmacophore model was utilized to identify small molecules that are AD4-11091-like.

Type H AD4-11091-Like

One structure derived from the AD4-10381-like pharmacophore is “Type H” as follows:

In the above structure, R¹²² and R¹²⁶ of Formula (32) can be independently selected from hydrogen, alkoxy, trifluoromethyl or halogen. Preferably, R¹²² and R¹²⁶ of Formula (32) are independently selected from hydrogen, trifluoromethyl, methoxy, or chloro. More preferably, one or both of R¹²² and R¹²⁶ of Formula (32) is hydrogen. Alkoxy and halogen are as defined above.

R¹²³ and R¹²⁵ of Formula (32) can be independently selected from hydrogen, trifluoromethyl, halogen, lower alkyl, acyl or alkoxy. Preferably, R¹²³ and R¹²⁵ of Formula (32) independently selected from halogen or trifluoromethyl. Halogen, lower alkyl, acyl and alkoxy are as defined above.

R¹²⁴ of Formula (32) can be selected from hydrogen, lower alkyl, alkoxy, acyl or halogen. Preferably, R¹²⁴ of Formula (32) is lower alkyl or halogen. Lower alkyl, alkoxy, acyl and halogen are as defined above.

R¹²⁷ and R¹³¹ of Formula (32) can be independently selected from hydrogen, lower alkyl, or alkoxy. Preferably, R¹²⁷ and R¹³¹ of Formula (32) are independently selected from hydrogen, methyl, or methoxy. Lower alkyl and alkoxy are as defined above.

R¹²⁸ and R¹³⁰ of Formula (32) can be independently selected from hydrogen or halogen. Preferably, R¹²⁸ and R¹³⁰ of Formula (32) are independently selected from hydrogen or chloro. Halogen is as defined above.

R¹²⁹ of Formula (32) can be selected from hydrogen, halogen, alkoxy, or lower alkyl. Preferably, R¹²⁹ of Formula (32) is selected from hydrogen, chloro, methoxy, or methyl. Halogen, alkoxy, and lower alkyl are as defined above.

R¹³² of Formula (32) can be selected from hydrogen or lower alkyl. Preferably, R¹³² of Formula (32) is selected from hydrogen, methyl, or ethyl. Lower alkyl is as defined above.

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (32).

In some embodiments, the compound of Formula (32) is AD4-12509.

In some embodiments, the compound of Formula (32) is AD4-12522.

In some embodiments, the compound of Formula (32) is AD4-12528.

In some embodiments, the compound(s) of Formula (32) excludes compound AD4-11091, Formula (31).

Type I AD4-11091-Like

One structure derived from the AD4-10381-like pharmacophore is “Type I” as follows:

In the above structure, n of Formula (33) can be 1 or 2, which represents one or two methylene groups resulting in either a five or six membered carbocyclic ring.

R¹³³ and R¹³⁷ of Formula (33) can be independently selected from hydrogen, lower alkyl or halogen. Preferably, R¹³³ and R¹³⁷ of Formula (33) are independently selected from hydrogen, methyl or chloro. Lower alkyl and halogen are as defined above.

R¹³⁴ and R¹³⁶ of Formula (33) can be independently selected from hydrogen, trifluoromethyl, or halogen. Preferably, R¹³⁴ and R¹³⁶ of Formula (33) are independently selected hydrogen, trifluoromethyl, or chloro. Halogen is as defined above.

R¹³⁵ of Formula (33) can be selected from hydrogen or halogen. Preferably, R¹³⁵ of Formula (33) is chloro. Halogen is as defined above.

R¹³⁸, R¹⁴⁰, and R¹⁴¹ of Formula (33) can be independently selected from hydrogen.

R¹³⁹ of Formula (33) can be selected from hydrogen or halogen. Preferably, R¹³⁹ of Formula (33) is selected from fluoro or hydrogen. Halogen is as defined above.

R¹⁴² of Formula (33) can be selected from hydrogen or lower alkyl. Preferably, R¹⁴² of Formula (33) is selected from hydrogen or methyl. Lower alkyl is as defined above.

In some embodiments, the compound(s) are the enantiomeric isomers of Formula (33).

In some embodiments, the compound of Formula (33) is AD4-12846.

In some embodiments, the compound of Formula (33) is AD4-12239.

In some embodiments, the compound(s) of Formula (33) excludes compound AD4-11091, Formula (31).

Synthesis

One aspect provides methods for the synthesis of compounds described herein (see Example 9).

An AD4-1734-like compound can be synthesized as follows:

An AD4-1886-like compound can be synthesized as follows:

An AD4-11091-like compound can be synthesized as follows:

An AD4-11511-like compound can be synthesized as follows:

Further methodology for synthesis of AD4-12632, which is an AD4-11511-like compound, is provided in Example 9.

The above reactions can include any condition or combination of conditions disclosed in Example 9.

Pharmaceutical Formulations

Embodiments of the compositions of the invention include pharmaceutical formulations of the various compounds described herein. A compound described herein can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. The compounds described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers and/or excipients as described in, for example, Remington's Pharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005). Such formulations will contain a therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulation should suit the mode of administration. The agents of use with the current invention can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents of the present invention and/or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophillic or other physical forces.

Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent and reduce dosage frequency. Controlled-release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects.

Combination with Kinase Inhibitors

Compounds described herein can be used with, or formulated with, known therapeutic compounds. Combination therapy is understood as a therapeutic regimen comprising, e.g., an anti-proliferative compound described herein and a second agent. An anti-proliferative compound and a second agent can be formulated for separate administration or may be formulated for administration together.

Compounds described herein can be combined with another anti-prolifative compound, such as the EGFR kinase inhibitors, Tykerb, Iressa, and Tarceva, or Erbitux, a humanized monoclonal antibody to the EGF receptor, to produce a greater therapeutic effect than either agent alone. As shown herein, when AD4 compounds were evaluated in a cell proliferation assay with Tykerb, Iressa, Tarceva or Erbitux, the effect of the combination of agents to inhibit cell proliferation was greater than the effect of any of the agents alone (see e.g., Example 8). Specifically, compounds described herein were evaluated with Tykerb, Iressa, Tarceva or Erbitux at a fixed concentration ratio, which was ascertained from the results of dose-response curves of each agent alone.

An compound described herein, such as an EGFR inhibitor, can be used with, or formulated and used with a second agent that inhibits vascularization of a tumor. Vascularization of a solid tumor generally refers to formation of blood vessels in a solid tumor. An agent that inhibits the vascularization of a tumor can inhibit vessel initiation, development, or maintenance leading to, for example, the reduction in the number or density of vessels in a tumor.

A compound described herein can be used with, or formulated and used with a second agent that modifies, for example increasing, permeability of a solid tumor. Permeability of a solid tumor generally refers to the permeability of a solid tumor to a therapeutic. A solid tumor may be said to be permeable to a therapeutic if the therapeutic is able to reach cells at the center of the tumor.

A compound described herein can be used with, or formulated and used with, a chemotherapeutic second agent. A chemotherapeutic agent refers to a molecule or composition used to treat a malignancy. Such agents can be used in combination with a compound described herein or with a combination therapy described herein. Chemotherapeutic agents include agents that can be conjugated to a compound described herein or can be used in combination with the combination therapy in unconjugated form.

A compound described herein can be used with, or formulated and used with a second agent that is a biological agent. A biological agent, also called a biologic, are generally understood as a product of a biological system, e.g., an organism, cell, or recombinant system. Examples of such biologic agents include, but are not limited to, nucleic acid molecules (e.g., antisense nucleic acid molecules), interferons, interleukins, colony-stimulating factors, antibodies (e.g., monoclonal antibodies), and cytokines.

A compound described herein can be used or formulated with an EGFR inhibitor approved for treatment of an EGFR-related condition or disorder. For example, compounds described herein can be used with or formulated with one or more of Tykerb, Iressa, Tarceva, or Erbitux. Tykerb, Iressa, and Tarceva are kinase inhibitors that block EGFR tyrosine kinase activity. Erbitux is a humanized monoclonal antibody that binds to an extracellular epitope on EGFR. Erbitux blocks activation of the receptor by preventing both ligand binding and receptor dimerization. In various embodiments, a compound described herein can lock EGFR into a dimerization incompetent conformation. Thus, compounds described herein and known EGFR inhibitors, such as those described above, can act in a complementary or synergistic fashion.

A compound described herein, such as AD4-1734-like compounds, AD4-1886-like compounds, AD4-10381-like compounds, or AD4-11091-like compounds, can be used or formulated with Tykerb. Compounds described herein, such as AD4-1734-like compounds, AD4-1886-like compounds, AD4-10381-like compounds, or AD4-11091-like compounds, can be used or formulated with Iressa. A compound described herein, such as AD4AD4-1734-like compounds, AD4-1886-like compounds, AD4-10381-like compounds, or AD4-11091-like compounds, can be used or formulated with or Tarceva.

Therapeutic Use

Another aspect is a process of treating a proliferative disease, disorder, or condition with a compound described herein. In various embodiments, a proliferative disease, disorder, or condition is associated with a target biomolecule having an interdomain tether associated with activation state, such as EGFR. The therapeutic method can include administration of a therapeutically effective amount of a compound of the invention to a subject in need thereof. In some embodiments, the compound is a compound described herein having anti-proliferative effects. In some embodiments, the compound is a compound described herein EGFR inhibitory activity. In some embodiments, the compound is an EGFR inhibitor that acts to bind one or more domains of EGFR so as to prevent tether extension and maintain an inactive conformation.

In various embodiments, the therapeutic method includes administration of one or more compounds described herein.

For example, the therapeutic method can include administration of one or more compounds according to a formula selected from the following: Formula (13); Formula (14); Formula (15); Formula (16); Formula (18); Formula (19); Formula (20); Formula (21); Formula (22); Formula (23); Formula (25); Formula (27); Formula (28); Formula (32); and Formula (33), as defined above, or a stereoisomer or pharmaceutically acceptable salt thereof.

As another example, the therapeutic method can include administration of one or more compounds selected from the following: AD4-1734 (Formula (12)); AD4-1886 (Formula (17)); AD4-10381 (Formula (24)); and AD4-11091 (Formula (31)), or a stereoisomer or pharmaceutically acceptable salt thereof.

As another example, the therapeutic method can include administration of one or more compounds selected from the following:

or a stereoisomer or pharmaceutically acceptable salt thereof.

Methods described herein are generally performed on a subject in need thereof. For example, a subject in need of the therapeutic methods described herein can be diagnosed with a proliferative disease, disorder, or condition, or at risk thereof. As another example, a subject in need of the therapeutic methods described herein can be diagnosed with a disease, disorder, or condition associated with EGFR, or at risk thereof. A determination of the need for treatment can be assessed by a history and physical exam consistent with the disease, disorder, or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, preferably a mammal, more preferably horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, guinea pigs, and chickens, and most preferably a human.

Examples of proliferative diseases or conditions treatable with compositions described herein include, but are not limited to, cancer; blood vessel proliferative disorders; fibrotic disorders; mesangial cell proliferative disorders; psoriasis; actinic keratoses; seborrheic keratoses; warts; keloid scars; eczema; and hyperproliferative diseases caused by virus infections, such as papilloma virus infection.

While under no obligation to provide an underlying mechanism and in no way limiting the present invention by doing so, it is presently thought that at least a portion of activity of compounds described herein arise from inhibition of EGFR. It is further contemplated that the presently described compounds may have additional modes of action in their effectiveness in treating a proliferative disease, disorder, or condition. Regardless of the underlying mechanism, compounds described herein have been demonstrated to be empirically effective in treating proliferative diseases and conditions.

Various compounds described herein can be effective for inhibiting EGFR, and thus, effective against diseases or conditions associated with EGFR, such as include, but are not limited to, proliferative diseases. In some embodiments, the proliferative disease treated by a compound described herein is a condition caused by excessive growth of cancer or non-cancer cells that express a member of the EGFR family of receptors. The excess cells generated by a proliferative disease can express EGFR at normal levels or can overexpress EGFR. Particularly suitable diseases or conditions associated with EGFR can be those stimulated by a ligand of EGFR or mutations of such ligands. Examples of such ligands that stimulate EGFR include, but are not limited to, EGF, TGF-alpha, heparin-binding growth factor (HBGF), β-cellulin, and Cripto-1. Examples of proliferative disease associated with EGFR include, but are not limited to, cancer; blood vessel proliferative disorders; fibrotic disorders; mesangial cell proliferative disorders; psoriasis; actinic keratoses; seborrheic keratoses; warts; keloid scars; eczema; and hyperproliferative diseases caused by virus infections, such as papilloma virus infection.

Cancer, or neoplasia, refers generally to any malignant neoplasm or spontaneous growth or proliferation of cells. A subject having “cancer”, for example, may have a leukemia, lymphoma, or other malignancy of blood cells. In certain embodiments, the subject methods are used to treat a solid tumor. Exemplary solid tumors include but are not limited to non small cell lung cancer (NSCLC), testicular cancer, lung cancer, ovarian cancer, uterine cancer, cervical cancer, pancreatic cancer, colorectal cancer (CRC), breast cancer, as well as prostate, gastric, skin, stomach, esophageal, and bladder cancer.

Treatment of cancer or treating a subject having cancer can include inhibition of replication of cancer cells, inhibition of spread of cancer, reduction in tumor size, lessening or reducing the number of cancerous cells in the body of a subject, or amelioration or alleviation of symptoms of cancer. A treatment can be considered therapeutic if there is a decrease in mortality or morbidity, and can be performed prophylactically, or therapeutically.

Methods described herein can be used to treat (e.g., reduce tumor size, decrease the vascularization, and/or increase the permeability of) an established tumor. An established tumor is generally understood as a solid tumor of sufficient size such that nutrients, e.g., oxygen, can no longer permeate to the center of the tumor from the subject's vasculature by osmosis and therefore the tumor requires its own vascular supply to receive nutrients. Methods described herein can be used to treat a solid tumor that is not quiescent and is actively undergoing exponential growth.

A therapeutic protocol can be modified according to permeability of a solid tumor. Permeability of a solid tumor generally refers to the permeability of a solid tumor to a therapeutic. A solid tumor may be said to be permeable to a therapeutic if the therapeutic is able to reach cells at the center of the tumor. An agent that increases the permeability of a tumor may for example, normalize, e.g., maintain, the vasculature of a solid tumor. Tumor vascularization or tumor permeability can be determined by a variety of methods known in the art, such as, e.g. by immunohistochemical analysis of biopsy specimens, or by imaging techniques, such as sonography of the tumor, computed tomography (CT) or magnetic resonance imaging (MRI) scans.

Different types of psoriasis display characteristics such as pus-like blisters (pustular psoriasis), severe sloughing of the skin (erythrodermic psoriasis), drop-like dots (guttae psoriasis) and smooth inflamed lesions (inverse psoriasis). The treatment of all types of psoriasis (e.g., psoriasis vulgaris, psoriasis pustulosa, psoriasis erythrodermica, psoriasis arthropathica, parapsoriasis, palmoplantar pustulosis) is contemplated by the invention.

Blood vessel proliferative disorders refer to angiogenic and vasculogenic disorders generally resulting in abnormal proliferation of blood vessels. The formation and spreading of blood vessels, or vasculogenesis and angiogenesis, respectively, play important roles in a variety of physiological processes such as embryonic development, corpus luteum formation, wound healing, and organ regeneration. They also play a pivotal role in cancer development. Other examples of blood vessel proliferation disorders include, but are not limited to, arthritis, where new capillary blood vessels invade the joint and destroy cartilage, and ocular diseases, like diabetic retinopathy, where new capillaries in the retina invade the vitreous, bleed and cause blindness. Disorders related to the shrinkage, contraction or closing of blood vessels, such as restenosis, are also implicated.

Fibrotic disorders refer to the abnormal formation of extracellular matrix. Examples of fibrotic disorders include, but are not limited to, hepatic cirrhosis and mesangial cell proliferative disorders. Hepatic cirrhosis is characterized by the increase in extracellular matrix constituents resulting in the formation of a hepatic scar. Hepatic cirrhosis can cause diseases such as cirrhosis of the liver. An increased extracellular matrix resulting in a hepatic scar can also be caused by viral infection such as hepatitis. Lipocytes appear to play a major role in hepatic cirrhosis. Other fibrotic disorders implicated include atherosclerosis.

Mesangial cell proliferative disorders refer to disorders brought about by abnormal proliferation of mesangial cells. Mesangial proliferative disorders include, but are not limited to, various human renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies.

EGFR (Tuzi et al., 1991, Br. J. Cancer 63:227-233; Torp et al., 1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989, Science 244:707-712) and the PDGF-R (Kumabe et al., 1992, Oncogene 7:627-633) are known to be overexpressed in many tumors and/or persistently activated by autocrine loops. Overexpression of the receptor and autocrine loops have been demonstrated in most common and severe cancers (see e.g., Akbasak and Suner-Akbasak et al., 1992, J. Neurol. Sci. 111:119-133; Dickson et al., 1992, Cancer Treatment Res. 61:249-273; Korc et al., 1992, J. Clin. Invest. 90:1352-1360; Lee and Donoghue, 1992, J. Cell. Biol. 118:1057-1070). Overexpression of EGFR is known to be associated with cancers of the bladder, brain, head and neck, pancreas, lung, breast, ovary, colon, prostate, and kidney. (see e.g., Atalay et al., 2003, Ann. Oncology 14:1346-1363; Herbst and Shin, 2002, Cancer 94:1593-1611; Modjtahedi et al., 1996, Br. J. Cancer 73:228-235). Overexpression of EGFR can be correlated or associated with poor prognosis of the patients (see e.g., Herbst and Shin, 2002, Cancer 94:1593-1611; Modjtahedi et al., 1996, Br. J. Cancer 73:228-235). HER2 has been associated with breast, ovarian, gastric, lung, pancreas and bladder cancer.

An inhibitor compound described herein can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery to within or to other organs in the body.

According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

An effective amount of a compound described herein is generally that which can exhibit an anti-proliferative effect to an extent such as to ameliorate the treated condition. For example, an effective amount of a compound described herein may inhibit EGFR to an extent such as to ameliorate the treated condition. In some embodiments, an effective amount is that amount of therapy (or combination therapy) that is sufficient to affect a desired result on a cancerous cell or tumor, including, but not limited to, for example, reducing tumor size, reducing tumor volume, decreasing vascularization of a solid tumor, or increasing the permeability of a solid tumor to an agent, either in vitro or in vivo. In certain embodiments, an effective amount of therapy (or combination therapy) is the amount that results in a percent tumor inhibition of more than about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%. In certain embodiments, an effective amount of therapy (or combination therapy) is sufficient to achieve a desired clinical result, including but not limited to, for example, ameliorating disease, stabilizing a subject, preventing or delaying the development of, or progression of cancer in a subject. An effective amount of therapy (or combination therapy) can be determined based on one administration or repeated administration. Methods of detection and measurement of the indicators above are known to those of skill in the art. Such methods include, but are not limited to measuring reduction in tumor burden, reduction of tumor size, reduction of tumor volume, reduction in proliferation of secondary tumors, decreased solid tumor vascularization, expression of genes in tumor tissue, presence of biomarkers, lymph node involvement, histologic grade, and nuclear grade.

In some embodiments, tumor burden can be determined. Tumor burden, also referred to as tumor load, generally refers to a total amount of tumor material distributed throughout the body of a subject. Tumor burden can refer to a total number of cancer cells or a total size of tumor(s), throughout the body, including lymph nodes and bone barrow. Tumor burden can be determined by a variety of methods known in the art, such as, for example, by measuring the dimensions of tumor(s) upon removal from the subject, e.g., using calipers, or while in the body using imaging techniques, e.g., ultrasound, computed tomography (CT) or magnetic resonance imaging (MRI) scans. Tumor size can be determined, for example, by determining tumor weight or tumor volume.

When used in the methods of the invention, a therapeutically effective amount of a compound described herein can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the agents of the invention can be administered, at a reasonable benefit/risk ratio applicable in a sufficient amount sufficient to inhibit the target biomolecule for which the compound is specific for the treatment or prophylaxis of a disease, disorder, or condition associated with the target biomolecule.

Toxicity and therapeutic efficacy of such compounds, and pharmaceutical formulations thereof, can be determined by standard pharmaceutical procedures in cell cultures and/or experimental animals for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD₅₀/ED₅₀, where large therapeutic indices are preferred.

The amount of a compound described herein may be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

Administration of a compound described herein can occur as a single event, a periodic event, or over a time course of treatment. For example, modulators can be administered daily, weekly, bi-weekly, or monthly. As another example, a compound can be adminstered in multiple treatment sessions, such as 2 weeks on, 2 weeks off, and then repeated twice; or every 3rd day for 3 weeks. One of ordinary skill will understand these regimes to be exemplary and could design other suitable periodic regimes. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4^(th) ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by an attending physician within the scope of sound medical judgment.

Compounds of the invention that inhibit a target biomolecule can also be used in combination with other therapeutic modalities. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for particular conditions linked to the target biomolecule. Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for a disease, disorder, or condition associated with a target biomolecule for which the compound is specific.

Kits

Also provided are kits. Such kits can include the compositions of the present invention and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein, for example, treatment methodologies or screening methodologies. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to one or more compounds described herein, vectors, diagnostic reagents, assay reagents, and/or combinations thereof. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, or sterile saline, each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustrate the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the invention, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 EGFR Inhibition Assay

The following example describes general EGFR in-cell Western (ICW) screening protocol. Methods are according to Chen et al. (2005) Analytical Biochemistry 338, 136-142 (incorporated herein by reference), except as otherwise noted.

Cell Plates: A431 cells (ATCC # CRL-1555) were grown in Dulbecco's Modified Eagle Medium (DMEM; ATCC #30-2002) supplemented with 10% fetal bovine serum, 105 units/ml pen/strep (Invitrogen #15140155) and 2.1 mM L-glutamine (ATCC #30-2214). Cells were seeded into 96 well tissue culture plates (BD Falcon #353948) at a density of 30,000 cells per well and incubated at 37° C. with 5% CO₂ overnight. Two cell plates were prepared for each compound plate.

Serum Starvation: The cells were serum starved prior to compound addition. The media was removed by aspiration and the cells washed with PBS (200 μl/well; Invitrogen #20012-027). The PBS was removed by aspiration and replaced with 200 μl of DMEM (ATCC #30-2002) supplemented with 105 units/ml pen/strep (Invitrogen #15140155) and 2.1 mM 1-glutamine (ATCC #30-2214). The cell plates were incubated at 37° C./5% CO₂ for 2 hours.

Compound Plate Preparation: The test compounds were solvated in 100% DMSO (Sigma #472301-2L) at a concentration of 25 mM. Compounds found not to be completely soluble in 100% DMSO at 25 mM were diluted to 10 mM with 100% DMSO and TFA (Fluka #91699) added to a final concentration of 0.2%. 40 μl of test compound was added to the appropriate well of a 96 well plate (Falcon #351190). As controls, 6 μM EGFR kinase inhibitor PD168393 (EMD/Calbiochem #513033) in 100% DMSO and DMSO alone were added to various wells. Prepared compound plates were stored at RT prior to use and at 4° C. long term.

Compound Dilution Plate Preparation: 250 μl of DMEM supplemented with 1 mg/ml BSA (Sigma A3059-10G) was added to the appropriate wells of a 96 well plate to prepare the compound dilution plates. Using a multichannel pipettor, 1.25 μl of compound from the compound plate was transferred into the compound dilution plate. This dilution rate will give a compound concentration in the assay of 125 μM.

Compound Addition: The starve media was removed from the cell plates by aspiration. Using a multichannel pipettor, the compound dilution plates were mixed by pipetting up and down three times. 50 μl of mixed, diluted compound was added to each of two rows/columns on each of two cell plates. The cell plates with compound were incubated at 37° C./5% CO₂ for 4 hours.

EGF Addition: 20 ng/ml EGF (Upstate #01-107) was prepared in DMEM supplemented with 1 mg/ml BSA. 50 μl of 20 or 0 ng/ml EGF was added to the appropriate wells without the removal of compound. The compound and EGF were mixed by pipetting up and down three times. The plates were incubated at 37° C./5% CO₂ for 10 min. In some screening assays the final concentration of EGF used for simulation was 6.6, or 12.5 ng/ml rather than 10 ng/ml.

Fixation and Triton Washing: The EGF+compound was removed by aspiration and 150 μl of freshly prepared Fixation Solution (1×PBS, Sigma P3813-10PK, and 4% Formaldehyde, Pierce #28908) was immediately added. The plates were incubated at RT for 20 min without shaking. The Fixation Solution was removed by aspiration and the plates washed four times with 200 μl each of Triton Wash Solution (1×PBS, Sigma P3813-10PK, and 0.1% Triton X-100, T8787-50ML) for 5 min with gentle shaking.

Blocking and Probing: Following the last Triton Wash, the plates were blocked for 1.5 h at RT with shaking using 150 μl of Odyssey Blocking Buffer (LI-COR #927-40000). The block was removed by aspiration and 50 μl of diluted primary Ab mix was added. The plates were incubated at 4° C. overnight with gentle shaking. The plates were washed five times with 200 μl of PBST (1×PBS, Sigma P3813-10PK, and 0.1% Tween-20, Sigma P9416-50ML) for 5 min each with shaking 50 μl of diluted secondary Ab mix was added and the plates incubated at RT for 1 h with shaking. The plates were washed 5× with 200 μl of PBST for 5 min each with shaking. One final wash with 200 μl of PBS (Sigma P3813-10PK) for 5 min with shaking was performed prior to scanning.

Primary Ab Mix contained: 0.1% Tween-20 (Sigma P9416-50ML); 1/500 dilution anti-total EGFR (Invitrogen #AHR5062); 1/800 dilution anti-phospho EGFR (Tyr 1173; Cell Signalling #4407); and Odyssey Blocking Buffer (LI-COR #927-40000). Some screening assays used a 1/100 dilution of anti-phospho EGFR (Tyr1045; Cell Signalling #2237) rather than the anti-phospho EGFR Ab indicated above.

Secondary Ab Mix contained: 0.2% 10% Tween-20 (Sigma P9416-50ML); 1/1200 dilution anti-mouse IR680 conjugate (LI-COR 926-32220); 1/1200 dilution anti-rabbit IR800CW conjugate (LI-COR 926-32211); and Odyssey Blocking Buffer (LI-COR #927-40000). In some screening assays, the dilution of the secondary Ab conjugates was 1/800 rather than 1/1200.

Plate Scanning. The plates were scanned on an Odyssey Infrared Imaging System from LI-COR Biosciences. The focus offset was set at 3.5 mm and the scanning intensity set at 3 for the 700 channel and 7 for the 800 channel.

Data Analysis: The % of Maximum and % Inhibition values were calculated as follows. 700 Channel=signal for total EGFR (used to control for variation in cell number). 800 Channel=signal for phosphorylated EGFR. 800_(−EGF)=basal level EGFR phosphorylation+non-specific signal (no compound). 800_(+EGF)=800_(−EGF)+EGF dependent EGFR receptor phosphorylation (no compound). 700/800_(com)=700 or 800 channel signals with compound. % Maximum={[(800_(com)/700_(com))−(800_(−EGF)/700_(−EGF))]/[(800_(+EGF)/700_(+EGF))−(800_(−EGF)/700_(−EGF))]}×100%. % Inhibition=100%−(% Maximum).

Example 2 MTT Assay

The following example describes the MTT Cell Proliferation Assay. The MTT Cell Proliferation Assay served as a secondary screen to evaluate active compounds from the primary cell based ICW screening protocol, described above. The MTT assay was used to evaluate toxicity through viability and proliferation effects, and compares the growth of an epithelial carcinoma A431 cell line (American Type Culture Collections (ATCC) cat #CRL-155) and an MDBK line (ATCC cat #CCL-22) derived from a healthy bovine kidney after a three day compound treatment and incubation.

The MTT Cell Proliferation Assay is a colorimetric assay system that measures the reduction of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into insoluble formazan crystals, produced by the mitochondria of viable cells. After incubation of the cells with the MTT, the formazan crystals formed are solubilized by the addition of a detergent or DMSO (ACS Reagent grade, Sigma cat#472301). The color can then be quantified by spectrophotometric means. Among the applications for the method are drug sensitivity, cytotoxicity, response to growth factors, and cell activation. The reduction of tetrazolium salts is now recognized as a safe, accurate alternative to radiometric testing.

Methods are according to manufacturer instructions for MTT Cell Proliferation Assay, ATCC, Cat. No. 30-1010K, except as otherwise noted.

Each MTT assay tested 11 compounds and 1 standard in a dose response curve with 5 concentrations, and 2.5 fold dilutions. Two replicates on each cell plate and 2 duplicate plates gave 4 individual replicates of each concentration, in each cell line. Concentrations were diluted in 100% DMSO from 25 mM stocks. Initial concentrations used and final concentrations tested were as follows: 20 mM to 100 μM; 8 mM to 40 μM; 3.2 mM to 16 μM; 1.3 mM to 6.4 μM; and 0.51 mM to 2.6 μM.

Twelve 96 well plates of each cell line were plated. Plating was done by dislodging cells as in passage protocol, counting on a hemocytometer, re-suspending in standard growth media, and plating using a multichannel pippetor.

Standards: AD4-10289 was used as the dose response standard, run at one concentration lower than the test compounds; 40, 16, 6.4, 2.6, and 1.0 μM final concentrations. 10289 was run in place of 1 test compound, with the same number of replicates. 16 μl of 25 mM stock+34 μl DMSO=8 mM starting concentrations, and serial diluted as with test compounds in same plate. AD4-1734 was used at 2 concentrations, 31.125 μM and 23.5 μM final concentrations. These concentrations were used on each plate to give 100%, and less than 100% activity in A431, while also giving less than 50% and 0 activity in DMBK cells. Two replicates of each concentrations was run on each cell plate. All compound addition and cell treatments were done using aseptic technique under a laminar flow aseptic hood.

Control: 0.5% DMSO in treatment media was used as the control. The assay was demonstrated to tolerate up to 1.0% DMSO with no significant growth differences.

MTT dye (Thiazolyl Blue Tetrazolium Bromide, Sigma cat #M5655) stock was prepared in bulk and stored at 4° C. for use: 5 mgs/ml sterile PDS, shielded from light. 4 ul of this stock per 100 uls treatment media per well was used. 130 ml of MTT/treatment was prepared fresh to dye 12 plates; 124.8 ml treatment media+522 mls of MTT stock.

Cells (harvested from 95% confluent flasks) were plated at densities of 7500 cells per well. Plates were incubated overnight prior to treatment, with treatment day=Day 0. Compound treatments were prepared prior to washing cells to minimize time cells were dry at room temperature after final aspiration. Compounds were diluted in 100% DMSO in 96 well polypropylene plates: 40 μl 25 mM stock+10 μl DMSO=20 mM; 20 μl of 20 mM+30 μl DMSO=8 mM; 20 μl of 8 mM+30 μl DMSO=3.2 mM; 20 μl of 3.2 mM+30 μl DMSO=1.3 mM; 20 μl of 1.3 mM+30 μl DMSO=0.51 mM.

5.0 μl/ml were added to treatment media, in sterile, 2.2 ml deep well plates=0.5% dmso final. Ten μl of each dmso concentration was added to 2.0 ml of treatment media and then mixed using a 1200 μl multichannel pipettor. Cell plates were treated from the deep well plates. Treatment media was standard growth media containing 0.5% FBS and standard additions. This reduced FBS concentration allowed for a slower growth rate in cells. In plate 1 of each cell line, standard AD4-10289 was run in lane 3 (see above for rates).

Media was aspirated from cell plates, and washed with 200 μl/well sterile PBS. Aspirated PBS wash, and cell plates were treated with 200 μl of compound/treatment media per well, and incubated at 37° C., 5% CO₂ for 3 days.

Prior to harvesting, visual observations were made under an inverted microscope. Plates were harvested by: aspirating the growth media; adding back MTT dye/100 μl treatment media; and incubating plates at 37° C. for 1 hour. MTT dye was aspirated. 100 μl/well dmso was added, and then shaken on a Bellco plate shaker for 5 minutes, at 4.5 setting.

Plates were read in a Tecan Sunrize UV plate reader, at 560 nm. Settings: Read mode, Outside Normal 2 sec; Shake settle time 3 sec. Data is reported as % inhibition, calculated for individual plates using control values from that plate.

Example 3 Testing of Identified Compounds from Pharmacophores for EGFR Inhibition

Identified compounds, representing various pharmacophore models, were tested for ability to inhibit EGFR at 25 μM.

AD4-compounds were identified using pharmacophore models (see Example 3, Example 4) and then were docked with the binding site of EGFR (SEQ ID NO: 1) that is recognized by defined CDRs of cetuximab. The inhibition of epidermal growth factor binding by AD4-compounds was then determined (NovaScreen BioSciences, Hanover, Md.). Inhibition of EGF binding was determined at 25 μM concentration.

For the inhibitor assays, K_(D) (binding affinity) was 1.04 nM, while B_(max) (receptor number) was 43.0 fmol/mg tissue (wet weight). Receptor source was rat liver membranes. The radioligand was [¹²⁵I]EGF (150-200 Ci/ng) at a final ligand concentration of 0.36 nM. A non-specific determinant was used as EGF—[100 nM]. The reference compound and positive control was EGF. Reactions were carried out in 10 mM HEPES (pH 7.4) containing 0.1% BSA at 25° C. for 60 minutes. The reaction was terminated by rapid vacuum filtration onto glass fiber filters. Radioactivity trapped onto the filters was determined and compared to control values to ascertain any interactions of test compounds with the EGF binding site. The EGF inhibitor assays were modified from, for example, Mukku (1984) J. Biol. Chem. 259, 6543-6546; Duh et al. (1990) World J. Surgery 14, 410-418; Lokeshwar et al. (1989) J. Biol. Chem. 264(32), 19318-19326.

Example 4 Generation of Pharmacophores for Target Inhibition from EGFR/Cetuximab Crystal Structure

The following example describes analysis of the target protein-antibody crystal structure and generation of pharmacophores for EGFR inhibition.

The protein crystal structure of cetuximab complexed to EGFR has been reported by Ferguson et al. (Cancer Cell, 2005, 7, 301-311 and the crystallographic data deposited in the Protein Data Bank as PDB code 1YY9. Structural information which define the position of the atoms of cetuximab was utilized to construct a pharmacophore model used to identify small molecules having similar atoms in similar positions. Small molecules having similar features to the antibody can demonstrate similar biological activity and thus similar therapeutic utility.

The pharmacophore feature generation and pharmacophore virtual screening module of the Molecular Operating Environment (MOE) software from Chemical Computing Group (CCG) (Montreal, Quebec, Canada) was used in the pharmacophore definitions described below. MOE's pharmacophore applications use a general notion of a pharmacophore being a set of structural features in a ligand that are directly related to the ligand's recognition at a receptor site and its biological activity.

In MOE, pharmacophoric structural features are represented by labeled points in space. Each ligand is assigned an annotation, which is a set of structural features that may contribute to the ligand's pharmacophore. A database of annotated ligands can be searched with a query that represents a pharmacophore hypothesis. The result of such a search is a set of matches that align the pharmacophoric features of the query to the pharmacophoric features present in the ligands of the searched database. The MOE software suite provides for interactive modifications (positions, radii, as well as other characteristics of the pharmacophoric query can be interactively adjusted); systematic matching (all possible matches of the ligand and the query are systematically examined); partial matching (the search algorithm is capable of finding ligands that match only a portion of the query); and volume filtering (the query can be focused by adding restrictions on the shape of the matched ligands in the form of a set of volumes).

The pharmacophore features of this example were generated using the Pharmacophore Query Editor in MOE. All hydrogen bond donor features are spheres of 1.2 Angstroms in radius and are colored purple. All hydrogen bond acceptor features are spheres of 1.2 Angstroms in radius and are colored cyan. All aromatic features are spheres of 1.2 Angstroms in radius and are colored green. All combined acceptor-anion pharmacophore features are spheres of 1.2 Angstroms in radius and are colored grey. All combined donor-acceptor features are spheres of 1.2 Angstroms in radius and are colored pink. All combined donor-cation features are spheres of 1.2 Angstroms and are colored red. All donor, acceptor, aromatic, combined acid-anion, and combined donor-acceptor directionality features are spheres of 1.5 Angstroms in radius and colored dark grey for donors, dark cyan for acceptors, dark green for aromatics, dark cyan for combined acid-anions, and dark grey for combined donor-acceptors. A feature that is marked essential in the pharmacophore query must be contained in the ligand in order for that ligand to be a hit.

All of the pharmacophore features were derived from the corresponding donor, acceptor, aromatic and acid moieties of the corresponding antibody in complex with its receptor (e.g., cetuximab complexed with EGFr, pdb accession number 1YY9) taken from crystal structures deposited in the protein databank with two exceptions. In some cases two methods provided the MOE software are used to place pharmacophore features. These are explained below.

The Contact statistics calculated, using the 3D atomic coordinates of a receptor, preferred locations for hydrophobic and hydrophilic ligand atoms using statistical methods. Using this method hydrophobic-aromatic and H-bonding features were placed, as noted in the individual pharmacophore definitions.

The MultiFragment Search essentially places a relatively large number of copies of a fragment (e.g., 200 copies of ethane) into a receptor's active site. The fragments are placed randomly around the active site atoms and are assumed not to interact with each other; no regard is paid to fragment overlap. Next, a special energy minimization protocol is used to refine the initial placement: the receptor atoms feel the average forces of the fragments, while each fragment feels the full force of the receptor but not of the other fragments. Using this technique it was possible to place hydrophobic, H-bond donors, acceptors and anions and cations in favorable positions within the receptors for use as MOE pharmacophore features.

Excluded volumes were generated for the pharmacophores defined below except when indicated. These were derived from the position of the receptor atoms near the antibody binding site. Excluded volumes are positions in space where ligand atoms must be excluded in order to avoid bumping into the receptor. They were generated in MOE by selecting the receptor residues within 5 Angstroms from the antibody and selecting “union” from the pharmacophore query editor in MOE.

In the Individual Pharmacophore Definitions described below, abbreviations were as follows: F=pharmacophore feature; Donor=Don, Acceptor=Acc, Anion=Ani, Cation=Cat, Acceptor and Anion=Acc&Ani, Donor and Cation=Don&Cat, Donor and Acceptor=Don&Acc, Aromatic=Aro, Hydrophobe=Hyd.

EGFR Complexed with Antibody Cetuximab (1YY9.Pdb)

The crystal (1YY9.pdb) of protein EGFR (SEQ ID NO: 1) complexed with antibody cetuximab (SEQ ID NO: 5 and SEQ ID NO:6) was analyzed according to the procedures described above. Results showed that two sets of residues of the antibody cetuximab make contact with the receptor. These are Gly54-Asp58 and Thr100-Glu105.

Hit AD4-1886 from Pharmacophore Model 21_Thr100_Glu105

Pharmacophore model 21_thr100_glu105 (described below in Table 1 and 2 and depicted in FIG. 1A-FIG. 1B) produced the hit, AD4-1886.

In the Individual Pharmacophore Definitions described below, abbreviations were as follows: F=pharmacophore feature; Donor=Don, Acceptor=Acc, Anion=Ani, Cation=Cat, Acceptor and Anion=Acc&Ani, Donor and Cation=Don&Cat, Donor and Acceptor=Don&Acc, Aromatic=Aro, Hydrophobe=Hyd.

TABLE 1 21_thr100_glu105 F(III)1 Derived from the side chain OH of Partial match, antibody Tyr102. This OH donates ligand must an H-bond to the side chain match at least 7 carbonyl of receptor pharmacophore Don&Acc Gln408 and accepts an H-bond from features. the side chain NH2 of receptor FIG. 1A-FIG. 1B Gln384. F(III)2 Derived from the backbone carbonyl Acc of antibody Tyr102 accepting an H-bond from the side chain OH of receptor Ser440. F(III)3 Directionality of F2 with respect Acc2 to the OH of Ser440. F(III)4 Derived from side chain phenol ring Aro of the antibody Tyr101 forming a favorable coulombic interaction with the imidazole side chain of the receptor His409. F(III)5 Directionality of F4 with respect Aro2 to the imidazole of His409. F(III)6 Derived from the side chain Acc&Ani carboxylate of antibody Asp103. Acceptor accepts an H-bond from or Anion forms a salt bridge to NH3+ of receptor Lys465. F(III)7 Directionality of F6 with respect Acc2 to the side chain of Lys465. F(III)8 Derived from the side chain OH of Don antibody Tyr102. This OH donates an H-bond to the side chain carbonyl of receptor Gln408. This feature is marked essential. F(III)9 Directionality with respect to the Don2 side chain carbonyl of receptor Gln408. V1 Excluded volume

TABLE 2 Cartesian and spherical coordinates of features for pharmacophore 21_thr100_glu105. sphere sphere Θ Φ radius volume Preferred Feature X Y Z r (theta) (phi) (A) (A³) features F(III)1 37.771 38.261 61.320 81.552 41.243 45.369 1.2 7.2 Don&Acc F(III)2 35.242 44.754 66.137 87.287 40.739 51.781 1.2 7.2 Acc F(III)3 35.245 45.639 64.234 86.320 41.915 52.323 1.5 14.1 Acc2 F(III)4 39.910 35.496 67.237 85.870 38.463 41.650 1.2 7.2 Aro F(III)5 35.866 34.818 65.306 82.241 37.431 44.151 1.5 14.1 Aro2 F(III)6 31.030 44.156 69.520 88.009 37.822 54.903 1.2 7.2 Acc&Ani F(III)7 30.168 42.269 69.193 86.513 36.889 54.484 1.5 14.1 Acc2 F(III)8 37.771 38.261 61.320 81.552 41.243 45.369 1.2 7.2 preferred Don F(III)9 37.200 36.592 60.180 79.652 40.928 44.528 1.6 17.2 Don2

Hit AD4-11091 from Pharmacophore Model Pharm-1886-6

Pharmacophore model Pharm-1886-6, which was derived from the Glide docked pose of AD4-1886, afforded the hit AD4-11091. This is a partial match model. The ligand must match at least 9 pharmacophore features recited in Table 3 and 4.

TABLE 3 Pharm-1886-6 F(V)1 Ligand accepts an H-bond from side chain Partial match, Acc&Ani amino of receptor Lys465 ligand must F(V)2 Directionality of F1 with respect to the match at least 9 Acc2 side chain amino of receptor Lys465 pharmacophore F(V)3 Directionality of F1 with respect to the features. Acc2 side chain amino of receptor Lys465 FIG. 2 F(V)4 Ligand donates an H-bond to the side Don chain carbonyl of receptor Gln411. This feature is marked essential F(V)5 Directionality of F4 with respect to the Don2 side chain carbonyl of Gln411 F(V)6 Ligand donates an H-bond to the side Don chain nitrogen of receptor Gln411. This feature is marked essential. F(V)7 Directionality of F6 with respect to Don2 the side chain nitrogen of Gln411 F(V)8 Ligand accepts an H-bond to the side Acc chain imidazole of receptor His409 F(V)9 Ligand forms a favorable π-π interaction Aro with the phenyl ring of receptor Phe412 F(V)10 Directionality of F9 with respect to the Aro2 phenyl ring of receptor Phe412 F(V)11 Ligand forms favorable hydrophobic Aro interactions with the side chains of receptor Val417 and Ile448. This feature is marked essential V1 Excluded volume

TABLE 4 Cartesian and spherical coordinates of features for pharmacophore Pharm-1886-6. sphere sphere Θ Φ radius volume Preferred Feature X Y Z r (theta) (phi) (A) (A³) features F(V)1 29.158 39.252 69.930 85.329 34.962 53.394 1.2 7.2 Acc&Ani F(V)2 30.366 40.690 70.870 87.180 35.618 53.267 1.5 14.1 Acc2 F(V)3 27.798 40.331 71.112 86.349 34.560 55.424 1.5 14.1 Acc2 F(V)4 30.334 37.857 68.525 83.958 35.296 51.296 1.2 7.2 preferred Don F(V)5 30.012 36.233 67.233 82.060 34.984 50.365 1.5 14.1 Don2 F(V)6 32.328 37.488 67.721 83.884 36.166 49.227 1.2 7.2 preferred Don F(V)7 31.212 36.002 66.743 82.006 35.523 49.076 1.5 14.1 Don2 F(V)8 38.730 36.298 66.517 85.100 38.590 43.143 1.2 7.2 Acc F(V)9 35.061 37.556 66.983 84.418 37.489 46.968 1.2 7.2 Aro F(V)10 34.495 37.311 64.976 82.486 38.027 47.246 1.5 14.1 Aro2 F(V)11 36.483 41.320 63.964 84.438 40.753 48.557 1.2 7.2 preferred Aro

Using the above described methodology, one can generate pharmacophore models for a variety of protein targets (crystallized with ligand).

Example 5 Generation of Pharmacophores for Target Inhibition from Inactive EGFR Crystal Structure

The following example describes analysis of the target protein crystal structure and generation of pharmacophores for EGFR inhibition. The ligands found by this methodology will interact with residues from Dom II and Dom IV of EGFR and thereby yielding an inactive form of the receptor.

Methods are according to those described in Example 4, except as noted otherwise. Abbreviations for Individual Pharmacophore Definitions are as described above.

The protein crystal structure of the inactive conformation of EGFR has been reported by Ferguson et al. (Ferguson, K. M., Berger, M. B., Mendrola, J. M., Cho, H., Leahy, D. J., Lemmon, M. A. (2003) EGF activates its receptor by removing interactions that auto-inhibit ectodomain dimerization Mol. Cell. 11: 507-517). The binding site was determined using the site finder module in the MOE software. It consists of the interface of residues from Domain II (23 residues, Lys227, Phe228, Lys 235, Asp236, Thr237, Cys238, Pro239, Pro240, Leu241, Met242, Tyr244, Tyr249, Gln250, Met251, Gly257, Lys258, Tyr259, Ser260, Cys265, Val275, His278, Gly279 and Ser280) and Domain IV (16 residues, Arg548, Gly549, Pro550, Asp551, Asn552, Asp561, His564, Val566, Thr568, Cys569, Pro570, Ala571, Gly572, Val573, Met574 and Leu580) (see e.g., FIG. 3).

Structural information which was derived from contact statistics and MFSS in the program MOE was used to construct pharmacophore models used to identify small molecules having similar atoms in similar positions.

Two methods provided in the MOE software are used to place pharmacophore features which correspond to hit AD4-1734. These are contact statistics and MFSS, as described above. Both the contact statistics and MFSS algorithms were applied to the Domain II-Domain IV interface binding site of the inactive form of EGFr (1NQL.pdb, see figure above).

Excluded volumes were generated in MOE by selecting the receptor residues of the binding site at the Domain II and Domain IV interface described above and selecting “union” from the pharmacophore query editor in MOE.

In the Individual Pharmacophore Definitions described below, abbreviations were as follows: F=pharmacophore feature; Donor=Don, Acceptor=Acc, Anion=Ani, Cation=Cat, Acceptor and Anion=Acc&Ani, Donor and Cation=Don&Cat, Donor and Acceptor=Don&Acc, Aromatic=Aro, Hydrophobe=Hyd.

Hit AD4-1734 from Pharmacophore Model Pharm-1nql-Glue-6

Pharmacophore model Pharm-1 nql-glue-6 (see TABLE 5 and 8; FIG. 4) produced the hit AD4-1734. This is a partial match model. The ligand must match at least 5 pharmacophore features.

TABLE 5 Pharm-1nql-glue-6 F(II)1 Derived from MFSS (see above). Partial match, Don&Cat Ligand donates an H-bond or forms a ligand must salt bridge to the carboxylate side match at least 5 chain of receptor Asp553 pharmacophore F(II)2 Derived from MFSS. This feature is features. Don ignored in this pharmacophore model FIG. 4 F(II)3 Derived from hydrophobic contact Hyd statistics. Ligand forms hydrophobic contacts with side chain of receptor Val568, imidazolidine side chain of receptor Pro552 and with the side chain of Met253 F(II)4 Derived from hydrophobic contact Hyd statistics. Ligand forms hydrophobic contacts with side chain of receptor Val575, Met253, and with the imidazolidine ring of receptor Pro552. This feature is marked essential F(II)5 Derived from MFSS. Ligand donates an Don H-bond to the side chain hydroxyl of Thr570 F(II)6 Directionality of F4 with respect to Don2 the backbone carbonyl of receptor Thr570 F(II)7 Derived from MFSS. Ligand accepts an Acc H-bond from receptor backbone NH of Ala573 F(II)8 Directionality of F7 with respect to Acc2 the backbone NH of receptor Ala573 F(II)9 Derived from MFSS. Ligand donates an Don&Cat H-bond to the backbone carbonyl of Asp563 and forms a salt bridge to the side chain carboxylate of receptor Asp563. This feature is marked essential V1 Excluded volume

TABLE 6 Cartesian and spherical coordinates of features for pharmacophore Pharm-1nql-Glue-6. sphere sphere Θ Φ radius volume Preferred Feature X Y Z r (theta) (phi) (A) (A³) features F(II)1 53.523 −17.135 −4.293 56.363 94.368 −17.752 1.2 7.2 Don&Cat F(II)2 47.983 −20.609 −10.619 53.290 101.494 −23.244 1 4.2 optional Don F(II)3 50.511 −16.780 −7.319 53.726 97.830 −18.377 1.7 20.6 Hyd F(II)4 48.632 −18.658 −12.837 53.647 103.844 −20.990 1.4 11.5 preferred Hyd F(II)5 44.675 −21.359 −12.586 51.093 104.261 −25.552 1.2 7.2 Don F(II)6 45.067 −23.421 −12.644 52.340 103.980 −27.461 1.5 14.1 Don2 F(II)7 44.906 −20.294 −14.555 51.383 106.455 −24.319 1.2 7.2 Acc F(II)8 45.475 −21.640 −16.064 52.861 107.691 −25.448 1.5 14.1 Acc2 F(II)9 52.952 −20.952 −9.221 57.688 99.198 −21.588 1.2 7.2 preferred Don&Cat

Hit AD4-10381 from Pharmacophore Model DockPharm 1505-2

Pharmacophore model DockPharm 1505-2 (see TABLE 7 and TABLE 8; FIG. 5), afforded the hit AD4-10381. This is a partial match model. The ligand must match at least 8 pharmacophore features.

TABLE 7 DockPharm1505-2 F(IV)1 Derived from MFSS. Ligand accepts an Partial match, Acc H-bond from receptor side chain OH of ligand must Thr239. This feature is marked essential match at least 8 F(IV)2 Directionality of F1 with respect to pharmacophore Acc2 side chain OH of Thr239. This feature features. is marked essential FIG. 5 F(IV)3 Derived from MFSS. F Ligand forms Aro hydrophobic contacts with side chain of receptor Met576 and imidazolidine ring of Pro242 F(IV)4 Directionality of F3 with respect to Aro2 side chain of receptor Met576 and imidazilidine ring of Pro242 F(IV)5 Derived from MFSS. Ligand forms Aro hydrophobic contacts with side chain of receptor Met576 and imidazolidine ring of Pro242 F(IV)6 Directionality of F5 with respect to Aro2 side chain of receptor Met576 and imidazolidine ring of Pro242 F(IV)7 Derived from MFSS. Ligand forms Aro hydrophobic contacts with side chain of receptor Leu243 and side chain of Thr239 F(IV)8 Derived from MFSS. Ligand donates an Don H-bond to the backbone carbonyl of His280. This feature is marked essential F(IV)9 Derived from hydrophobic contact Aro statistics. Ligand forms hydrophobic contacts with side chains of receptor Met244 and Leu243. This feature is marked essential F(IV)10 Derived from MFSS. Ligand forms Hyd hydrophobic contacts with side chain of receptor Met244 V1 Excluded volume

TABLE 8 Cartesian and spherical coordinates of features for pharmacophore DockPharm 1505-2. sphere sphere Θ Φ radius volume Preferred Feature X Y Z r (theta) (phi) (A) (A³) features F(IV)1 43.406 −13.282 −20.203 49.686 113.993 −17.014 1.2 7.2 preferred Acc F(IV)2 41.702 −12.124 −20.610 48.071 115.388 −16.211 1.6 17.2 preferred Acc2 F(IV)3 44.536 −14.059 −19.940 50.781 113.121 −17.520 1.2 7.2 Aro F(IV)4 45.616 −12.975 −21.378 52.021 114.264 −15.878 1.5 14.1 Aro2 F(IV)5 45.210 −12.475 −18.241 50.322 111.253 −15.426 1.2 7.2 Aro F(IV)6 46.291 −11.393 −19.681 51.575 112.433 −13.827 1.5 14.1 Aro2 F(IV)7 43.427 −7.811 −18.296 47.767 112.521 −10.196 1.2 7.2 Aro F(IV)8 41.296 −6.448 −17.166 45.184 112.328 −8.875 1.2 7.2 preferred Don F(IV)9 46.569 −6.963 −15.306 49.512 108.007 −8.504 1.2 7.2 preferred Aro F(IV)10 48.308 −6.291 −13.040 50.431 104.985 −7.420 1.8 24.4 Hyd

Example 6 Ligand Docking and Scoring to EGFR Protein Complexed with Antibody Cetuximab

The compounds selected for docking to the target protein, crystal (1YY9.pdb) of protein EGFR (SEQ ID NO: 1) complexed with antibody cetuximab (SEQ ID NO: 5 and SEQ ID NO:6), were those which were found to align to the pharmacophore models generated in the MOE modeling software (see Example 3). These compounds were obtained in MOE database format. The 3-dimensional atomic coordinates of these compounds were written to a structure data format (*.sdf) file using the export command in the MOE database window without adding hydrogens.

The LigPrep software module of Maestro modeling software (Schrodinger LLC, NY, N.Y.) was next employed to prepare the compounds for docking. The *.sdf file was converted into Maestro format using LigPrep. Hydrogens were then added and any charged groups neutralized. Ionization states were generated for the ligands at 7.0+/−1.0 pH units. After this, tautomers were generated when necessary, alternate chiralities were generated and low energy ring conformers were produced. This was followed by removing any problematic structures and energy minimizing the resulting ligands using MacroModel software module. Finally a Maestro file (*.mae) was written of the ligands which were now ready for docking. All of these steps were automated via a python script supplied by Schrodinger, LLC.

The following describes protein preparation. First the protein structure 1 YY9 of EGFR complexed with antibody cetuximab was imported into Maestro in PDB format. Hydrogens were added and any errors such as incomplete residues were repaired. The protein structure was checked for metal ions and cofactors. Charges and atom types were set for metal ions and cofactors as needed. Ligand bond orders and formal charges were adjusted if necessary. The binding site was determined by picking the ligand (for 1YY9 it is either the Thr100-Tyr101-Tyr102-Asp103-Tyr104-Glu105 or Gly54-Gly55-Asn56-Thr57-Asp58 pieces of the antibody) in Maestro (Glide). The program determines the centroid of the picked ligand and draws a 20 Angstrom box which is the default setting with the centroid of the ligand at the center of the box. The box was the binding site for the ligands to be docked. The protein preparation facility, which is automated in Glide, consists of two components, preparation and refinement. The preparation component added hydrogens and neutralized side chains that are not close to the binding site and do not participate in salt bridges. The refinement component performed a restrained minimization of the co-crystallized complex which reoriented side-chain hydroxyl groups and alleviated potential steric clashes.

The following describes receptor grid generation. Glide searches for favorable interactions between one or more ligand molecules and a receptor molecule, usually a protein. The shape and properties of the receptor are represented on a grid by several different sets of fields including hydrogen bonding, coulombic (i.e., charge-charge) interactions hydrophobic interactions, and steric clashes of the ligand with the protein. In the first step the receptor must be defined. This was done by picking the ligand. The unpicked part of the structure was the receptor. The ligand was not included in the grid calculation but was used to define the binding site as described above. Scaling of the nonpolar atoms of the receptor was not included in the present docking runs. The grids themselves were calculated within the space of the enclosing box. This is the box described above and all of the ligand atoms must be contained in this box. No pharmacophore constraints were used because the Glide extra precision scoring function performs better without these constraints.

To use Glide, each ligand must be a single molecule, while the receptor may include more than one molecule, e.g., a protein and a cofactor. Glide can be run in rigid or flexible docking modes; the latter automatically generates conformations for each input ligand. The combination of position and orientation of a ligand relative to the receptor, along with its conformation in flexible docking, is referred to as a ligand pose. All docking runs are done using the flexible docking mode. The ligand poses that Glide generates pass through a series of hierarchical filters that evaluate the ligand's interaction with the receptor. The initial filters test the spatial fit of the ligand to the defined active site, and examine the complementarity of ligand-receptor interactions using a grid-based method. Poses that pass these initial screens enter the final stage of the algorithm, which involves evaluation and minimization of a grid approximation to the OPLS-AA nonbonded ligand-receptor interaction energy. Final scoring is then carried out on the energy-minimized poses. By default, Schrödinger's proprietary GlideScore multi-ligand scoring function is used to score the poses. If GlideScore was selected as the scoring function, a composite Emodel score is then used to rank the poses of each ligand and to select the poses to be reported to the user. Emodel combines GlideScore, the nonbonded interaction energy, and, for flexible docking, the excess internal energy of the generated ligand conformation. Conformational flexibility is handled in Glide by an extensive conformational search, augmented by a heuristic screen that rapidly eliminates unsuitable conformations, such as conformations that have long-range internal hydrogen bonds.

The settings used in the docking runs of this example were as follows. Grid file was read in. Extra precision (XP) scoring function was used. Docked using conformational flexibility. 5000 poses per ligand for the initial Glide screen were kept (default). Scoring window for keeping initial poses was 100.0 (default). Best 800 poses per ligand for the energy minimization was kept (default). For the energy minimization, a distance dependent dielectric constant of 2.0 was used and maximum number of conjugate gradient steps was 100 (defaults). The ligand file was then loaded. Molecules with >120 atoms and/or >20 rotatable bonds were not docked (default). Van der Waals radii of ligand atoms with partial charges <0.15 were scaled by 0.80. This was done to mimic receptor flexibility. Constraints and similarity were not used. Poses with Coulomb plus Van der Waals energies >0.0 were rejected. To ensure that poses for each molecule were conformationally distinct, poses with RMS deviation <0.5 and/or maximum atomic displacement of 1.3 Angstroms were discarded.

The following describes Glide Scoring. The choice of best-docked structure for each ligand was made using a model energy score (Emodel) that combines the energy grid score, the binding affinity predicted by GlideScore, and (for flexible docking) the internal strain energy for the model potential used to direct the conformational-search algorithm. Glide also computed a specially constructed Coulomb-van der Waals interaction-energy score (CvdW) that was formulated to avoid overly rewarding charge-charge interactions at the expense of charge-dipole and dipole-dipole interactions. This score was intended to be more suitable for comparing the binding affinities of different ligands than is the “raw” Coulomb-van der Waals interaction energy. In the final data work-up, one can combine the computed GlideScore and “modified” Coulomb-van der Waals score values to give a composite score that can help improve enrichment factors in database screening applications. The mathematical form of the Glide score is:

GScore=0.065*EvdW+0.130*Coul+Lipo+Hbond+Metal+BuryP+RotB+Site

where EvdW is van der Waals energy (calculated with reduced net ionic charges on groups with formal charges, such as metals, carboxylates, and guanidiniums); Coul is the Coulomb energy (calculated with reduced net ionic charges on groups with formal charges, such as metals, carboxylates, and guanidiniums); Lipo is the lipophilic contact term (rewards favorable hydrophobic interactions); HBond is the hydrogen-bonding term (separated into differently weighted components that depend on whether the donor and acceptor are neutral, one is neutral and the other is charged, or both are charged); metal is the metal-binding term (only the interactions with anionic acceptor atoms are included; if the net metal charge in the apo protein is positive, the preference for anionic ligands is included; if the net charge is zero, the preference is suppressed); BuryP is the penalty for buried polar groups; RotB is the penalty for freezing rotatable bonds; and Site is polar interactions in the active site (polar but non-hydrogen-bonding atoms in a hydrophobic region are rewarded).

The following describes generation of the virtual compound library that was screened. The lead-like compounds from a free, virtual database of commercially available compounds was downloaded in structure data format (sdf, Molecular Design Limited) from the ZINC database (Irwin and Shoichet (2005) J. Chem. Inf. Model. 45(1), 177-182). The lead-like database is comprised of approximately 890,000 compounds divided into 33 segments. This was used to generate the database of conformers for screening by MOE. Hydrogens were then added. For a pharmacophore search, a database of low energy conformers must be generated. The Conformation Import command was applied to the sdf file above. After the conformers were generated, preprocessing of the conformer database was applied. This step, called feature annotation, determined the types of pharmacophore features in each molecule/conformation and their geometrical relationships. This was then compared with the query and those molecules/conformations that matched the query within the given tolerance were saved as hits.

AD4-1886-Like

Analysis of compounds from the ZINC database against pharmacophore 21_thr100_glu105 identified from the 1YY9.pdb crystal of protein EGFR (SEQ ID NO: 1) complexed with antibody cetuximab (SEQ ID NO: 5 and SEQ ID NO:6) (see e.g., Example 3) according to the methods described above identified the compound AD4-1886.

The compounds in TABLE 9 were identified via AD4-1886 structure similarity searches and were docked to the 1YY9.pdb binding site (erbitux residues thr100_glu105) to obtain their Glide and Emodel scores. Also depicted in TABLE 9 is ICW assay and MTT assay results (see Example 1, Example 2, Example 7).

TABLE 9 Glide Score, E-Model score, ICW assay, and MTT Assay Results for AD4-1886-like Compounds. Glue Glue- Erbitux- Erbitux- Sub- G- E- ICW MTT G- E- AD4-1886 by ICW Inhibition Class Score Model Screen Screen Score Model

AD4- 11883 O −6.72 −56.32 83.13 19.51/ non- conver- gent −3.22 −50.29

AD4- 11975 K −5.19 −59.024 75.826 17.52/ 17.16 (0.98) −5.78 −36.79

AD4- 11409 K −5.51 −61.783 74.94 17.18/ 11.66 (0.68) −2.96 −34.87

AD4- 11638 O −5.68 −63.037 68.754 11.19/ 14.90 (1.33) −3.24 −50.39

AD4- 11645 O −7.29 −61.027 66.452 45.60/ 47.02 (1.03) −3.56 −39.45

AD4- 10608 L −6.31 −65.526 62.846 6.33/ 5.35 (1.18) −3.57 −38.88

AD4- 10676 K −4.99 −35.282 59.637 18.58/ 8.84 (0.48) −3.48 −39.56

AD4- 12119 K −4.87 −59.115 55.94 18.64/ 14.62 (0.78) −1.86 −39.07

AD4- 10706 K −5.17 −38.332 55.497 15.12/ 23.11 (1.53) −0.32 −35.65

AD4- 11416 K −5.11 −57.963 53.517 15.37/ 13.54 (0.88) −5.3 −41.96

AD4- 12148 O −5.18 −66.098 51.845 42.82/ 39.24 (0.92) −3.95 −43.35

AD4- 12152 K −6.63 −69.497 50.528 21.02/ 16.20 (0.77) −3.38 −44.49

AD4- 10626 M −7.03 −54.119 50.05 5.31/ 19.57 (3.68) −4.2 −40.24

AD4- 10624 L −5.71 −66.733 49.448 25.64/ 16.76 (0.65) −2.01 −33.65

AD4- 12097 K −7.24 −55.243 48.543 15.08/ 9.85 (0.65) −3.72 −36.73

AD4- 12040 K −5.67 −59.95 47.822 17.13/ 14.82 (0.87) −4.17 −33.64

AD4- 10705 K −4.28 −36.375 47.764 23.09/ 25.20 (1.09) −0.07 −35.1

AD4- 11367 O −6.03 −60.038 46.872 43.33/ 43.63 (1.01) −3.56 −32.87

AD4- 11413 K −5.42 −52.398 46.23 31.40/ 29.77 (0.95) 7 −4.38 −41.06

AD4- 10623 M −6.86 −51.962 46.185 25.78/ 38.34 (1.49) −4.83 −44.1

AD4- 11871 O −5.5 −63.42 45.776 49.72/ 41.84 (0.84) −2.83 −36.58

AD4- 12164 K −5.78 −50.998 45.64 50.58/ non- conver- gent −4.3 −45.59 51.37/ 549.1 (1.07)

AD4- 11968 K −7.13 −59.876 45.095 6.49/ 6.29 (0.97) −3.78 −36.75

AD4- 11422 K −5.81 −57.435 44.982 29.46/ 9.23 (0.31) −3.75 −38.46

AD4- 11971 K −5.84 −56.864 44.908 41.54/ 41.10 (0.99) −2.14 −36.33

AD4- 10609 L −5.54 −63.839 43.828 6.84/ 6.34 (0.93) −3.09 −40.73

AD4- 11369 O −5.24 −57.914 43.546 48.64/ 47.17 (0.97) −3.5 −43.41

AD4- 12041 L −5.79 −47.552 43.515 6.61/ 14.29 (2.16) −1.92 −34.81

AD4- 11970 N −5.62 −44.739 43.246 non- conver- gent −0.26 −35.75

AD4- 12175 K −3.1 −60.769 42.36 39.99/ 9.56 (0.24) −3.36 −29.19

AD4- 12107 N −6.12 −53.813 41.933 14.08/ non- conver- gent −3.53 −38.84

AD4- 12280 K −4.17 −55.921 41.439 10.23/ 17.68 (1.73) −1.38 −41.29 5.76/ 21.24 (3.69)

AD4- 12139 K −4.11 −63.237 41.23 18.56/ 5.30 (0.29) −5.93 −41.9

AD4- 12104 N −5.48 −48.445 41.204 38.98/ 37.55 (1.30) −4.1 −36.8

AD4- 11642 O −6.28 −65.521 40.922 9.89/ 9.46 (0.96) −3.16 −44.18

AD4- 12146 N −2.65 −54.642 40.916 26.57/ 25.76 (0.97) −3.96 −31.29

AD4- 11417 K −5.19 −59.747 40.711 25.47/ 49.29 (1.94) −4.49 −46.82

AD4- 11985 L −5.94 −48.648 40.346 62.51/ 39.85 (0.64) −3.44 −30.5

AD4- 11419 K −5.01 −63.607 40.289 31.32/ 38.81 (1.24) −2.95 −44.3

AD4- 12262 O −5.64 −55.994 40.146 42.85/ 43.44 (1.01) −3.46 −49.03

AD4- 10120 L −6.73 −62.371 39.873 22.84/ 39.27 (1.72) −4.84 −37.89

AD4- 11979 L −4.83 −50.876 39.775 9.31/ 16.16 (1.74) −2.24 −30.07

AD4- 11648 O −5.95 −54.648 38.362 46.74/ 45.07 (0.96) −3.43 −42.24

AD4- 10611 L −6.72 −63.131 38.285 30.94/ 20.04 (0.65) −2.77 −42.62

AD4- 11646 O −5.63 −58.436 37.949 19.43/ 21.11 (1.09) −3.11 −43.16

AD4- 12126 L −5.72 −62.255 37.795 14.90/ 41.85 (2.81) −2.57 −35.52

AD4- 12183 K −3.58 −64.491 37.695 25.56/ 17.25 (0.46) −2.96 −34.21

AD4- 11880 O −4.5 −62.253 37.604 47.28/ 47.33 (1.00) −2.65 −45.91

AD4- 12171 L −5.28 −49.354 37.375 38.92/ 37.13 (0.95) −4.18 −43.51

AD4- 10708 K −5.35 −35.677 37.233 16.64/ 35.92 (2.16) −4 −36.76

AD4- 11421 N −5.75 −53.081 37.181 41.46/ 43.51 (1.05) −3.85 −34.1

AD4- 12049 L −5.44 −48.47 36.966 32.27/ 11.96 (0.37) −2.43 −24.87

AD4- 12158 J −6.58 −71.788 36.727 5.01/ 4.58 (0.91) −4.16 −49.79

AD4- 12165 K −6.81 −66.767 36.632 63.62/ 12.03 (0.19) −3.94 −47.61

AD4- 11415 L −6.25 −70.703 35.841 15.74/ 10.52 (0.67) −4.75 −47.34

AD4- 12288 K −5.92 −54.007 35.584 non- conver- gent −4.78 −40.58

AD4- 10613 L −5.8 −59.786 35.514 13.92/ 11.84 (0.85) −3.36 −37.53

AD4- 12173 O −4.83 −49.002 35.393 37.26/ 105.8 (2.84) −2.76 −34.16 55.76/ 54.00 (0.97)

AD4- 12122 O −5.82 −52.564 35.068 non- conver- gent/ 40.29 −2.89 −44.52

AD4- 11538 L −6.95 −65.791 34.978 5.48/ 5.19 (0.95) −3.36 −40.64

AD4- 12064 N −4.99 −54.071 34.778 47.78/ 42.73 (0.89) −2.6 −34.85

AD4- 12042 K −5.83 −56.582 34.592 non- conver- gent −2.07 −29.6

AD4- 12016 L −4.01 −54.12 34.399 15.78/ 23.53 (1.49) −2.74 −30.33

AD4- 12196 K −6.07 −70.658 34.379 18.01/ 36.80 (2.04) −1.74 −49.22

AD4- 12044 K −5.61 −54.634 34.334 63.18/ 33.11 (0.52) −2.09 −37.5

AD4- 12060 N −5.79 −59.463 34.272 48.53/ 40.47 (0.83) −3.38 −36.21

AD4- 12062 K −6.61 −55.772 34.268 64.51/ 18.83 (0.29) −3.49 −36.09

AD4- 11881 O −6.34 −65.317 34.08 63.31/ 46.45 (0.73) −3.58 −44.41

AD4- 11418 K −4.89 −66.222 34.067 11.91/ 10.25 (0.86) −3.64 −51.66

AD4- 12010 K −7.06 −56.321 33.772 6.68/ 6.16 (0.92) −3.62 −37.4

AD4- 11402 N −4.4 −60.537 33.635 11.82/ 17.36 (1.47) −3.56 −43.59

AD4- 11977 N −4.93 −42.763 33.588 32.51/ non- conver- gent −3.11 −28.88

AD4- 11637 O −5.26 −60.687 33.491 non- conver- gent/ 17.76 −3.04 −46.33

AD4- 12149 K −5.38 −67.463 33.331 21.88/ 36.21 (0.60) −2.96 −49.52

AD4- 12273 O −4.61 −53.119 33.104 31.74/ 42.94 (1.35) −2.59 −34.17

AD4- 11874 O −6.4 −52.192 32.474 34.98/ 38.97 (1.11) −3.3 −48.79

AD4- 11867 O −4.91 −55.985 32.432 51.01/ 48.09 (0.94) −3.11 −43.51

AD4- 11885 O −5.44 −52.547 32.38 37.83/ 48.34 (1.28) −2.88 −40.04

AD4- 12003 K −5.54 −53.098 32.007 11.53/ 12.79 (1.11) −4.01 −31.62

AD4- 11412 K −6.18 −58.012 31.958 11.42/ 20.02 (1.75) −4.65 −40.44

AD4- 12153 N −6.74 −58.542 31.36 15.58/ 21.77 (0.72) −3.2 −49.88

AD4- 12024 K −5.06 −59.351 31.244 6.77/ 6.34 (0.97) −3.51 −37.04

AD4- 12265 N −5.64 −51.979 31.19 38.12/ 35.06 (0.92) −4.14 −42.09

AD4- 11870 O −6.19 −57.173 30.849 55.07/ 47.30 (0.86) −3.16 −44.1

AD4- 10612 L −6.39 −64.392 30.841 11.68/ 13.21 (1.13) −1.5 −37.91

AD4- 10313 L −2.31 −36.062 30.774 20.85/ 17.62 (0.85) −3.5 −38.52

AD4- 11396 K −5.9 −61.408 30.745 25.09/ 21.23 (0.85) −4.36 −46.45

AD4- 12145 N −6.3 −51.616 30.469 40.50/ 41.75 (1.03) −1.35 −41.29

AD4- 12029 K −5.31 −45.509 30.376 25.32/ 51.15 (2.02) −3.9 −42.48

AD4- 12267 J −5.05 −43.093 30.36 non- conver- gent −3.71 −30.79

AD4- 11861 O −6.6 −58.006 30.242 −2.03 −43.96

AD4- 11420 N −4.8 −54.408 30.052 −3.02 −32.21

AD4- 12039 L −6.45 −54.185 29.977 −3.16 −35.07

AD4- 12074 K −5.08 −55.471 29.545 16.24/ 32.28 (1.99) −2.98 −39.72

AD4- 12093 K −6.31 −46.102 29.436 non- conver- gent −2.96 −39.97

AD4- 12073 L −5.45 −59.824 29.313 19.54/ 32.28 (1.65) −5.06 −41.61 11.59/ 16.97 (1.46)

AD4- 10103 M −5.14 −48.394 29.295 −4.54 −44.4

AD4- 12266 J −5.78 −68.303 29.289 49.64/ 45.88 (0.92) −4.54 −53.86

AD4- 11385 J −5.17 −53.196 28.892 −2.89 −29.47

AD4- 12051 L −5.4 −55.888 28.852 −3.34 −31.94

AD4- 12168 K −6 −59.724 28.819 non- conver- gent −3.48 −33.83 60.47/ 19.68 (0.33)

AD4- 11424 N −5.82 −59.614 28.422 −3.64 −28.82

AD4- 12059 N −4.54 −58.82 28.33 32.25/ 37.69 (1.17) −3.42 −40.97

AD4- 11384 J −5.62 −60.184 28.127 −3.68 −47.04

AD4- 11410 L −5.68 −61.822 28.101 −3.84 −40.3

AD4- 12071 L −4.56 −58.755 27.713 −4.19 −41.48

AD4- 11407 K −5.89 −52.032 27.585 −3.55 −37.01

AD4- 11536 J −6.57 −53.881 27.431 −3.46 −46.82

AD4- 12017 K −6.1 −52.279 27.373 6.53/ 5.93 (0.91) −5.41 −35.72

AD4- 11863 O −5.54 −59.966 27.36 −1.92 −39.12

AD4- 11994 K −6.23 −55.137 27.18 −1.85 −35.39

AD4- 11973 N −4.87 −45.457 27.176 −3.5 −42.87

AD4- 12147 N −3 −57.789 26.967 −0.46 −40.6

AD4- 11976 L −2.46 −54.259 26.918 −2.33 −34.3

AD4- 11406 N −7.04 −54.126 26.851 −4.09 −39.79

AD4- 12286 N −4.81 −52.846 26.829 15.46/ 29.94 (1.94) −4.26 −32.26

AD4- 12157 L −4.38 −52.787 26.73 −1.28 −39.47

AD4- 12163 J −6.08 −59.91 26.671 −3.35 −43.82

AD4- 11405 J −6.94 −58.643 26.497 −4.41 −53.71

AD4- 11888 O −6.74 −63.413 26.355 −3.37 −41.13

AD4- 11395 J −6.11 −55.753 26.301 −4.63 −49.78

AD4- 12103 K −7.21 −63.637 26.212 −3.1 −44.09

AD4- 11523 K −6 −59.162 26.098 −3.83 −33.07

AD4- 12075 L −6.25 −65.453 25.956 19.24/ 40.11 (2.08) −7.04 −41.76

AD4- 12098 K −5.89 −53.46 25.946 23.36/ 16.41 (0.70) 0.121 −33.78

AD4- 11879 O −4.9 −68.161 25.689 −2.48 −47.6

AD4- 12014 K −5.59 −65.343 25.611 −3.5 −34.96

AD4- 11401 N −5.59 −56.827 25.557 −3.9 −51.43

AD4- 11978 L −3.01 −51.532 25.53 −2.15 −31.77

AD4- 11986 L −5.62 −53.997 25.368 −4.32 −35.72

AD4- 10622 L −5.32 −69.992 25.347 −5 −49.48

AD4- 11980 J −4.48 −57.693 25.235 49.82/ 24.40 (0.49) −2.39 −48.62

AD4- 12291 M −7.05 −58.86 25.191 −3.47 −34.09

AD4- 12133 L −5.78 −42.682 25.058 −0.57 −35.65

AD4- 12092 L −4.77 −52.761 25.019 −1.58 −29.14

AD4-11091-Like

The AD4-11091-like compounds in TABLE 10 were identified via Glide docked pose of AD4-1886. Depicted in TABLE 10 is Glide score, E-model score, and ICW assay and MTT assay results (See Examples 1-3).

TABLE 10 Glide Score, E-Model score, ICW assay, and MTT Assay Results for AD4-11091-like Compounds. Glue- Glue- Erbitux Erbitux AD4- G- E- ICW -G- -E- Pharmacophore AD4-11091 LIKE by ICW Inhibition No. Score Model assay MTT assay Score Model Query

AD4- 12509 −6.15 −54.56 140.975 5.49/5.24(0.95) −2.169 −37.161 1YY9-AD4- 11091 LIKE

AD4- 12423 −5.16 −62.7 129.155 6.35/5.77(0.91) 4.23/2.92(0.69) −4.008 −31.651 1YY9-AD4- 11091 LIKE

AD4- 12528 −7.06 −54.89 123.946 5.43/5.30(0.98) −4.226 −40.6 1YY9-AD4- 11091 LIKE

AD4- 12522 −5.62 −59.43 123.893 6.20/5.33(0.86) −4.336 −39.519 1YY9-AD4- 11091 LIKE

AD4- 12851 −6.66 −56.44 118.637 6.51/5.39(0.83) −3.534 −35.395 1YY9-AD4- 11091 LIKE

AD4- 12504 −5.98 −54.77 118.46 11.09/15.61(1.41) −3.478 −32.583 1YY9-AD4- 11091 LIKE

AD4- 12835 −4.47 −54.41 116.579 6.15/5.89(0.96) −2.989 −24.087 1YY9-AD4- 11091 LIKE

AD4- 12833 −4.64 −56.3 115.634 5.94/5.96(1.00) −3.931 −36.628 1YY9-AD4- 11091 LIKE

AD4- 12494 −5.78 −61.9 112.965 5.64/5.71(1.01) −3.005 −37.074 1YY9-AD4- 11091 LIKE

AD4- 11091 −4.2 −43.59 112.858 non-convergent −3.987 −42.271 1NQL-AD4- 1734 top 3

AD4- 12844 −5.23 −51.72 112.768 6.80/6.13(0.90) −1.121 −36.357 1YY9-AD4- 11091 LIKE

AD4- 12436 −6.93 −61.5 108.826 5.47/5.53(1.01) −3.738 −34.38 1YY9-AD4- 11091 LIKE

AD4- 12505 −5.92 −59.81 93.3284 5.69/6.02(1.06) −3.807 −37.664 1YY9-AD4- 11091 LIKE

AD4- 12487 −6.35 −56.12 84.5602 6.45/5.50(0.85) −3.209 −35.185 1YY9-AD4- 11091 LIKE

AD4- 12424 −6.87 −61.52 82.3761 4.08/4.73(1.16) −3.929 −44.784 1YY9-AD4- 11091 LIKE

AD4- 12430 −5.64 −61.33 82.0877 4.07/5.54(1.36) −4.089 −37.858 1YY9-AD4- 11091 LIKE

AD4- 12842 −5.06 −58.74 80.8965 6.72/5.55(0.83) −4.203 −41.175 1YY9-AD4- 11091 LIKE

AD4- 12503 −6.81 −57.7 79.4624 4.05/5.43(1.34) −4.398 −46.889 1YY9-AD4- 11091 LIKE

AD4- 12442 −5.97 −64.21 79.1185 5.59/5.76(1.03) −3.929 −36.195 1YY9-AD4- 11091 LIKE

AD4- 12468 −6.39 −57.69 77.7928 4.04/5.41(1.34) −3.531 −39.573 1YY9-AD4- 11091 LIKE

AD4- 12389 −5.83 −54.1 72.5659 12.15/6.07(0.50) −1.372 −36.929 1YY9-AD4- 11091 LIKE

AD4- 12846 −4.89 −53.74 71.4924 71% at 2.6 microMolar 5.55/5.38(0.97) −4.09 −40.727 1YY9-AD4- 11091 LIKE

AD4- 12501 −6.53 −53.93 71.3823 4.06/4.06(1.00) −4.39 −49.596 1YY9-AD4- 11091 LIKE

AD4- 12400 −6.67 −56.3 69.4793 4.05/5.77(1.42) −4.487 −36.418 1YY9-AD4- 11091 LIKE

AD4- 12239 −4.79 −57.44 67.8291 58% at 2.6 microMolar 16.13/13.77(0.85) −3.787 −33.019 1YY9-AD4- 11091 LIKE

AD4- 12472 −4.86 −50.57 64.2647 6.23/5.42(0.87) −4.241 −30.519 1YY9-AD4- 11091 LIKE

AD4- 12529 −7.76 −56.15 63.9249 6.35/6.47(1.02) −4.728 −39.186 1YY9-AD4- 11091 LIKE

AD4- 12395 −4.85 −55.34 57.5206 7.92/6.96(0.88) −4.241 −44.652 1YY9-AD4- 11091 LIKE

AD4- 12783 −6.55 −55 57.4272 7.00/7.62(1.09) −4.334 −34.188 1YY9-AD4- 11091 LIKE

AD4- 12433 −5.66 −62.36 54.4095 29.06/38.50(1.32) −3.35 −37.559 1YY9-AD4- 11091 LIKE

AD4- 12438 −6.13 −61.5 53.245 5.69/5.90(1.04) −4.367 −31.989 1YY9-AD4- 11091 LIKE

AD4- 12461 −5.04 −52.58 53.1391 4.06/5.91(1.46) −3.308 −39.144 1YY9-AD4- 11091 LIKE

AD4- 10700 −6.44 −62.8 52.5996 14.69/14.35(0.98) −1.113 −36.75 1YY9-AD4- 1886 Dockpharm

AD4- 12822 −4.19 −52.61 51.4082 6.92/7.04(1.02) −3.072 −33.644 1YY9-AD4- 11091 LIKE

AD4- 12403 −5.67 −58.34 50.7196 4.03/5.30(1.32) −2.864 −39.727 1YY9-AD4- 11091 LIKE

AD4- 12798 −6.21 −53.39 49.531 4.74/5.69(1.20) −3.7 −41.52 1YY9-AD4- 11091 LIKE

AD4- 12820 −6.64 −59.05 47.471 5.98/5.93(0.99) −3.737 −43.758 1YY9-AD4- 11091 LIKE

AD4- 11432 −6.34 −60.05 47.0006 7.80/7.82(1.00) −2.138 −42.697 1YY9-AD4- 11091 LIKE

AD4- 12829 −4.49 −36.92 46.4407 36.97/37.27(1.01) −4.396 −24.523 1YY9-AD4- 11091 LIKE

AD4- 12836 −5.44 −57.16 46.1658 9.32/8.43(0.90) −4.633 −35.888 1YY9-AD4- 11091 LIKE

AD4- 12827 −6.59 −56.14 45.9206 6.49/6.40(0.99) −4.353 −37.272 1YY9-AD4- 11091 LIKE

AD4- 12521 −5.59 −54.87 45.7824 6.99/6.54(1.22) −3.596 −28.511 1YY9-AD4- 11091 LIKE

AD4- 11427 −6.36 −57.89 44.904 11.97/14.48(1.21) −3.173 −34.903 1YY9-AD4- 11091 LIKE

AD4- 12443 −5.91 −55.75 44.7896 6.71/6.91(1.03) −3.756 −40.035 1YY9-AD4- 11091 LIKE

AD4- 12477 −5.1 −56.94 44.2167 6.65/5.44(0.82) −4.704 −45.689 1YY9-AD4- 11091 LIKE

AD4- 12843 −4.42 −54.07 43.3347 74% at 2.6 microMolar 6.26/12.23(1.95) −3.616 −45.969 1YY9-AD4- 11091 LIKE

AD4- 12460 −6.63 −60.84 42.5115 6.33/5.61(0.87) −3.956 −34.204 1YY9-AD4- 11091 LIKE

AD4- 12560 −6.6 −67.91 42.3275 8.88/5.23(0.59) −2.493 −38.312 1YY9-AD4- 11091 LIKE

AD4- 12850 −4.74 −59.3 42.2628 31.78/35.77(1.13) −5.454 −36.419 1YY9-AD4- 11091 LIKE

AD4- 12481 −6.81 −56.97 42.006 10.38/11.04(1.06) −4.203 −42.388 1YY9-AD4- 11091 LIKE

AD4- 12435 −5.71 −52.17 41.4281 6.67/6.15(0.92) −3.596 −34.513 1YY9-AD4- 11091 LIKE

AD4- 12854 −6.57 −55.99 38.7055 6.64/8.19(1.23) −3.134 −34.386 1YY9-AD4- 11091 LIKE

AD4- 12246 −6.97 −57.98 38.3844 8.36/8.01(0.96) −3.234 −36.483 1YY9-AD4- 11091 LIKE

AD4- 12391 −6.13 −58.89 37.4191 11.09/9.09(0.82) −1.131 −37.349 1YY9-AD4- 11091 LIKE

AD4- 12853 −5.17 −52.29 36.92 8.76/7.51(0.86) −4.265 −38.827 1YY9-AD4- 11091 LIKE

AD4- 12413 −5.58 −54.77 35.1946 6.83/6.35(0.93) −4.789 −34.803 1YY9-AD4- 11091 LIKE

AD4- 12837 −6.48 −49.2 35.1542 6.28/5.68(0.90) −3.525 −37.042 1YY9-AD4- 11091 LIKE

AD4- 11344 −5.87 −60.29 34.1727 15.06/15.26(1.01) −2.857 −32.85 1YY9-AD4- 11091 LIKE

AD4- 12525 −6.67 −61.47 34.1311 14.75/12.15(0.82) −3.692 −39.056 1YY9-AD4- 11091 LIKE

AD4- 12411 −6.38 −56.96 34.0556 6.70/5.18(0.77) −3.267 −31.845 1YY9-AD4- 11091 LIKE

AD4- 12485 −5.32 −49.68 33.3993 6.98/7.19(1.03) −3.785 −35.542 1YY9-AD4- 11091 LIKE

AD4- 12828 −4.75 −55.94 32.6456 26.61/63.39(2.38) −4.427 −40.661 1YY9-AD4- 11091 LIKE

AD4- 12527 −6.11 −56.16 31.9587 26.91/20.01(0.74) −4.98 −40.481 1YY9-AD4- 11091 LIKE

AD4- 12517 −6.51 −58.67 31.5308 30.39/20.01(0.66) −3.452 −41.81 1YY9-AD4- 11091 LIKE

AD4- 12465 −5.59 −56.91 31.3476 9.92/6.54(0.66) −3.435 −35.487 1YY9-AD4- 11091 LIKE

AD4- 12823 −3.94 −58.13 30.8167 4.07/4.06(1.00) −1.408 −47.23 1YY9-AD4- 11091 LIKE

AD4- 12537 −6.29 −58.32 30.7051 35.37/38.65(1.09) −3.88 −38.727 1YY9-AD4- 11091 LIKE

AD4- 11428 −6.22 −59.58 29.8673 20.77/15.46(0.74) −3.692 −42.78 1YY9-AD4- 11091 LIKE

AD4- 12577 −6.85 −61.65 29.4151 6.62/5.92(0.89) −4.289 −30.521 1YY9-AD4- 11091 LIKE

AD4- 12250 −6.65 −62.41 29.3683 12.68/11.57(0.91) −3.033 −43.246 1YY9-AD4- 11091 LIKE

AD4- 12832 −3.94 −58.13 29.02 7.71/16.30(2.11) −1.408 −47.23 1YY9-AD4- 11091 LIKE

AD4- 12512 −5.28 −56.81 28.9126 6.40/5.83(0.91) −3.555 −34.118 1YY9-AD4- 11091 LIKE

AD4- 12482 −7.87 −59.36 28.3382 6.54/6.05(0.93) −5.834 −34.551 1YY9-AD4- 11091 LIKE

AD4- 11430 −6.25 −48.49 27.1482 19.83/16.84(0.85) −3.823 −33.209 1YY9-AD4- 11091 LIKE

AD4- 10693 −6.87 −60.55 26.8206 48.24/35.85(0.74) −2.528 −44.456 1YY9-AD4- 1886 Dockpharm

AD4- 12825 −5.72 −54.54 26.7866 6.78/5.46(0.81) −4.103 −37.363 1YY9-AD4- 11091 LIKE

AD4- 12431 −5.95 −50.11 26.7815 6.40/5.57(0.87) −4.12 −36.705 1YY9-AD4- 11091 LIKE

AD4- 12813 −6.36 −51.7 26.1431 11.76/13.63(1.16) −5.202 −33.055 1YY9-AD4- 11091 LIKE

AD4- 12499 −5.38 −58.36 26.0767 6.99/6.58(0.94) −2.372 −38.545 1YY9-AD4- 11091 LIKE

AD4- 12824 −4.66 −56.6 25.9501 Not Active −2.832 −37.478 1YY9-AD4- 11091 LIKE

AD4- 12502 −5.63 −60.71 25.1182 6.97/5.46(0.78) −2.992 −38.026 1YY9-AD4- 11091 LIKE

AD4- 12855 −4.57 −57.09 25.0542 6.83/6.25(0.92) −4.8 −33.063 1YY9-AD4- 11091 LIKE

Two-dimensional representations of the docked pose of AD4-1886, along with AD4-1886-like compounds were produced. Docking of compound AD4-1886 to EGFR is depicted, for example, in FIG. 6A. Docking of compound AD4-11883 (an AD4-1886-like compound) to EGFR is depicted, for example, in FIG. 6B. Docking of compound AD4-11975 (an AD4-1886-like compound) to EGFR is depicted, for example, in FIG. 6C. Docking of compound AD4-11409 (an AD4-1886-like compound) to EGFR is depicted, for example, in FIG. 6D. Docking of compound AD4-11638 (an AD4-1886-like compound) to EGFR is depicted, for example, in FIG. 6E. Docking of compound AD4-11645 (an AD4-1886-like compound) to EGFR is depicted, for example, in FIG. 6F.

Example 7 Ligand Docking and Scoring to Inactive EGFR Protein

The compounds selected for docking to the target protein, the inactive folded conformation of EGFr (PDB accession number 1NQL) were those which were found to align to the pharmacophore models generated in the MOE modeling software (see Example 4).

Methods are according to those described in Example 6, except as otherwise indicated.

The protein crystal structure of EGFr in its inactive state (1NQL.PBD) was imported into Maestro in PDB format. The binding site was determined by picking a ligand, ZINC3304802 which was one of the pharmacophore hits found by MOE, in Maestro (Glide).

Analysis of compounds from the ZINC database against the pharmacophores identified from the 1NQL.PDB crystal structure of protein EGFR according to the methods described above identified compounds AD4-1734, and AD4-10381.

The compounds in the following tables were identified via structure similarity searches and were docked to the binding site to obtain their Glide and Emodel scores.

AD4-1734-Like

The compounds in TABLE 11 were identified via AD4-1734 structure similarity searches and were docked to the 1NQL.PDB binding site to obtain their Glide and Emodel scores. Also depicted in TABLE 11 is ICW assay and MTT assay results (See Example 1, Example 2, Example 7).

TABLE 11 Glide Score, E-Model score, ICW assay, and MTT Assay Results for AD4-1734-like Compounds. Sub- E- G- ICW Pharmacophore AD4 No. Class Model Score Screen MTT Screen MTT Follow-up Query

AD4- 10631 P −4.999 −46.538 104.48 35.64/37.47 (1.05) 1NQL-Glue-AD4- 1734 LIKE

AD4-1734 R −6.985 −47.431 65.9 22.75/42.50 (1.87) 22.1/40.2(1.82) 1NQL-Glue

AD4- 10188 Q −6.118 −47.713 53.35 9.05/16.32 (1.80) 1NQL-Glue-AD4- 1734 LIKE

AD4- 10186 P −5.204 −49.183 47.518 40.10/45.74 (1.14) 1NQL-Glue-AD4- 1734 LIKE

AD4- 10633 P −5.019 −48.58 46.398 5.20/non- convergent Not Convergent 1NQL-Glue-AD4- 1734 LIKE

AD4- 10174 R −7.534 −48.44 45.85 2.26/8.58 (3.80) 4.42/101.2 (22.9) 1NQL-Glue-AD4- 1734 LIKE

AD4- 10639 S −5.635 −48.32 43.695 42.59/non-convergent 27% Max & −12% Max 1NQL-Glue-AD4- 1734 LIKE

AD4- 10176 Q −7.195 −47.579 42.066 50% Max & 11% Max 1NQL-Glue-AD4- 1734 LIKE

AD4- 10182 P −5.62 −40.619 39.51 6.78/43.31 (6.39) 86% Max & 15% Max 3.75/non- convergent 1NQL-Glue-AD4- 1734 LIKE

AD4- 10171 Q −6.276 −48.614 39.389 16.52/21.02(1.27) 1NQL-Glue-AD4- 1734 LIKE

AD4- 10178 Q −6.311 −41.019 34.907 22% Max & −19% Max 1NQL-Glue-AD4- 1734 LIKE

AD4- 10179 S −5.484 −53.221 34.742 13.16/13.87(1.05) 38% Max & 20% Max 1NQL-Glue-AD4- 1734 LIKE

AD4- 10181 R −5.427 −45.217 34.604 2.61/15.96 (6.11) 38% Max & 5% Max 1NQL-Glue-AD4- 1734 LIKE

AD4- 10180 P −6.42 −43.904 32.1 16.72/36.31(2.17) 70% Max & 13% Max 1NQL-Glue-AD4- 1734 LIKE

AD4- 10637 R −6.977 −48.57 30.057 20.81/41.33(1.99) 1NQL-Glue-AD4- 1734 LIKE

AD4- 10491 R −5.677 −45.143 29.223 22.22/40.61(1.83) 1NQL-Glue-AD4- 1734 LIKE

AD4- 10192 −5.691 −47.96 16.772 16.51/43.30 (2.62) 24% Max & 19% Max 1NQL-Glue-AD4- 1734 LIKE

AD4- 10217 −5.043 −45.23 −1.007 14.02/non-convergent 44% Max & 6% Max 1NQL-Glue-AD4- 1734 LIKE

AD4- 10618 −5.33 −55.695 −3.062 18.43/non-convergent 86% Max & 13% Max 1NQL-Glue-AD4- 1734 LIKE

AD4- 10209 −5.614 −42.711 −10.6 87% Max & 39% Max 2.50/50.15(20.0) 1NQL-Glue-AD4- 1734 LIKE

AD4- 10619 −6.255 −55.21 −44.55 87% @ 2.6 μM 2.97/3.06(1.03) 1NQL-Glue-AD4- 1734 LIKE

AD4-10381-Like

The compounds in TABLE 12 were identified via AD4-10381 structure similarity searches and were docked to the 1NQL.PDB binding site to obtain their Glide and Emodel scores. Also depicted in TABLE 12 is ICW assay and MTT assay results (See Example 1, Example 2, Example 7).

TABLE 12 Glide Score, E-Model score, ICW assay, and MTT Assay Results for AD4-10381-like Compounds. AD4 Sub- G- E- ICW ICW ICW MTT Assay MTT Follow- Structure No. Class Score Model 1 2 Avg IC₅₀(A431/MDBK) up

12632 D −5.83 −34.77 89.66 92.56 91.11 5.67/5.59(0.99)

12681 E −4.03 −45.89 89.93 89.02 89.48 15.63/25.40(1.63)

12732 D −5.1 −47.5 79.12 86.41 82.77 11.07/14.95(1.35)

10381 + TFA D −6.02 −50.11 87.04 78.48 82.76 2.10/3.01(1.43)

11511 D −4.58 −48.54 77.05 87.95 82.50 95% at 2.6 mM 1.26/0.57(0.45)

11462 D −5.28 −51.09 77.14 77.49 77.32 14.60/46.58(3.19)

11772 + TFA D −5.5 −43.52 80.68 73.50 77.09 9.75/9.04(0.93)

AD4- 12652 + TFA D −5.33 −42.7 82.35 64.55 73.45 NOT ACTIVE

AD4- 12641 + TFA D −5.24 −47.81 70.26 74.15 72.20 NOT ACTIVE

11484 + TFA F −5.62 −44.77 67.84 73.46 70.65 11.34/9.72(0.86)

12679 E −4.15 −50.62 73.98 67.01 70.50 15.14/42.02(2.78)

11741 D −3.89 −52.17 66.55 69.55 68.05 2.44/2.44 51.51/51.89 (1.01)

12723 + TFA D −5.93 −44 67.34 65.91 66.62 9.33/9.99(1.07)

11829 E −4.74 −36.8 62.56 68.79 65.68 19.57/16.18

12645 + TFA D −6.1 −43.6 60.00 69.95 64.97 6.49/6.29(0.97)

12735 + TFA D −5.8 −44.56 69.46 59.98 64.72 17.92/15.32(0.85)

11636 E −5.64 −48.26 61.28 63.84 62.56 26.75/14.70

11492 D −5.66 −48.59 53.35 71.23 62.29 non-convergent

12660 + TFA D −5.55 −47.79 49.87 73.40 61.63 41.54/41.10(0.99)

11445 + TFA D −4.56 −56.29 59.54 61.02 60.28 9.61/45.14(4.70)

11783 + TFA D −5.05 −44.32 66.95 53.44 60.19 18.22/14.99(0.82)

11436 + TFA F −4.46 −43.34 59.16 60.14 59.65 5.86/2.80(0.48)

11472 D −5.57 −43.39 51.74 66.90 59.32 24.68/19.35

12612 + TFA D −5.87 −46.47 69.21 45.51 57.36 48.51/79.16

12693 + TFA E −4.92 −42.18 40.36 74.00 57.18 9.01/6.88(0.76)

11812 E −4.52 −47.03 51.90 61.77 56.84 14.65/31.61

11601 D −6.11 −52.7 58.11 54.06 56.08 1.29/2.62 0.738/2.61(normal- ized) 3.67/48.56 (13.23)

11825 E −4.03 −50.59 52.17 55.87 54.02 4.06/17.94

12673 + TFA D −5.89 −44.35 44.70 60.90 52.80 33.08/22.52(0.68)

12613 D −5.37 −45.92 47.72 56.65 52.18 17.58/11.50(0.65)

11583 + TFA D −5.58 −53.92 47.93 54.26 51.10 47.28/47.33(1.00)

12604 + TFA D −3.38 −55.15 55.87 46.08 50.98 19.30/29.98(1.55)

11316 E −5.01 −42.35 47.95 53.57 50.76 44.42/19.89(0.45)

11450 D −2.82 −58.11 53.35 48.02 50.68 6.98/6.83

11582 D −6.23 −55.72 59.77 40.01 49.89 2.43/2.44 49.26/55.85 (1.13)

11315 E −4.77 −43 48.33 50.82 49.58 very selective MDBK 20% at 100 μM

11498 D −5.12 −47.66 42.15 56.48 49.32 5.39/8.77

12742 D −5.3 −45.02 33.49 64.92 49.20 19.60/18.95(0.97)

12636 E −4.58 −41.6 49.55 48.49 49.02 15.88/34.63(2.18)

11758 D −5.73 −43.23 53.63 41.93 47.78 53.81/576.2

11771 + TFA D −4.55 −44.55 51.58 42.32 46.95 18.17/42.13(2.32)

12611 + TFA D −5.46 −53.71 52.69 38.76 45.72 5.32/5.31(1.00)

12738 + TFA D −5.5 −46.21 53.67 37.02 45.35 45.36/24.62(0.54)

11753 + TFA D −5.13 −57.6 42.44 47.28 44.86 NOT ACTIVE

12665 + TFA D −5.51 −43.87 51.06 37.29 44.17 17.52/17.16(0.98)

11448 D −4.63 −45.4 50.06 37.38 43.72 3.45/11.8(3.27)

12736 D −5.39 −46.96 32.35 54.51 43.43 7.14/9.82(1.38)

11607 + TFA D −6.52 −43.34 43.69 41.73 42.71 10.08/8.03(0.80)

12608 + TFA D −5.19 −51.11 46.64 38.07 42.36 43.13/17.03(0.80)_

12737 D −5.3 −47.17 38.90 45.65 42.27 14.72/6.33(0.43)

12609 D −5.81 −43.86 42.02 40.07 41.04 11.19/23.49(2.10)

12683 + TFA D −5.91 −44.79 54.37 27.38 40.88 63.42/45.31(0.71)

12674 D −5.8 −34.96 29.69 51.69 40.69 10.67/8.41(0.79)

11707 + TFA D −4.94 −55.26 43.70 37.31 40.51 15.70/non-convergent

11474 D −4.35 −53.39 32.19 48.71 40.45 3.57/28.48(7.98)

11471 + TFA D −4.39 −57.49 37.43 42.40 39.92 36.91/44.14(1.20)

11712 + TFA D −4.74 −48.22 32.96 46.11 39.53 18.74/17.32(0.92)

11730 + TFA D −5.24 −46.64 47.15 30.67 38.91 14.48/10.05(0.69)

12226 + TFA D −5.67 −51.38 42.24 35.23 38.74 42.50/49.01(1.15)

12603 + TFA D not docked not docked 48.06 29.15 38.61 16.20/10.11(0.62)

11442 D −5.57 −51.14 50.14 25.76 37.95 15.63/118.5

12778 D −3.8 −52.22 31.05 44.08 37.56 4.66/5.34(1.15)

11455 E −5.64 −40.8 48.80 25.90 37.35 10.84/23.41(2.16)

11826 + TFA D −5.44 −59.4 40.52 33.80 37.16 49.43/43.30(0.88)

11449 E −3.52 −42.05 48.00 26.22 37.11 6.41/5.40(0.84)

11755 D −5.33 −64.06 40.96 33.21 37.09 non-convergent 14.14/3.24 (0.23)

11309 E −5.51 −42.35 33.31 40.70 37.00

12666 + TFA D −5.29 −49.45 48.16 25.37 36.77 14.48/17.13(1.18)

12688 + TFA E −5.44 −42.43 45.86 26.95 36.40 5.04/3.58(0.71)

12654 + TFA D −5.9 −44.23 37.92 34.22 36.07 15.95/12.62(0.79)

11742 + TFA D −5.95 −57.7 33.84 37.63 35.74 16.09/non-convergent

11456 D −5.61 −49.17 33.74 37.33 35.54 5.84/5.48(0.94)

11785 + TFA D −3.71 −54.57 41.06 30.01 35.54 10.35/9.15(0.88) 24.82/25.91 (1.04)

12661 D −4.69 −40.42 29.34 41.14 35.24 43.15/38.19(0.89)

12214 + TFA D −5.35 −48.28 42.29 27.52 34.91 32.43/45.47(1.40)

11706 + TFA F −1.8 −49.31 37.65 30.99 34.32 17.60/25.45(1.45)

12625 + TFA D −5.41 −45.41 30.45 38.10 34.27 21.20/17.03(0.80)

12739 D −5.22 −42.52 28.51 39.54 34.03 15.90/9.17(0.58)

11476 D −5.09 −57.08 36.71 31.11 33.91 17.86/13.29(0.74)

12741 + TFA D −5.68 −44.51 42.10 25.04 33.57 63.20/28.04(0.44)

11795 + TFA D −4.61 −48.34 35.23 31.81 33.52 26.80/12.87(0.48)

11773 + TFA D −5.37 −48.14 43.74 23.30 33.52 39.36/20.99(0.53)

11294 D −6.17 −45.01 29.81 37.13 33.47 6.43/19.78(3.08)

11513 + TFA D −5.96 −56.74 32.64 34.26 33.45 6.73/6.32(0.94)

11800 + TFA D −4.31 −58.11 34.26 32.02 33.14 6.71/5.86(0.87)

12620 D −4.98 −47.62 32.26 33.77 33.01 5.17/5.30(1.03)

11809 + TFA D −6.46 −44.94 31.42 34.02 32.72 39.28/10.88(0.28)

12746 D −5.22 −48.45 18.02 47.28 32.65 17.86/11.89(0.67)

12607 + TFA D −5.11 −44.98 38.40 26.85 32.62 16.57/11.83(0.71)

11759 + TFA D −5.06 −46.66 38.43 26.39 32.41 14.20/7.36(0.52)

11503 D −5.4 −50.16 25.88 38.53 32.21 6.71/19.02(2.83)

12667 D −5.45 −48.78 29.45 34.27 31.86 36.05/19.55(0.54)

12655 D −5.54 −42.42 30.00 33.36 31.68 11.59/7.89(0.68)

11610 F −5.54 −43.39 22.50 40.84 31.67 4.98/17.38

12610 + TFA D −5.54 −43.39 41.25 21.42 31.33 50.40/19.62(0.39)

11727 + TFA D −5.45 −54.36 37.00 25.09 31.05 35.11/16.14(0.46)

11726 + TFA D −5.33 −54.88 35.72 26.02 30.87 NOT ACTIVE

12777 D −5.54 −42.95 24.37 37.17 30.77 12.81/8.96(0.70)

12689 + TFA E −4.12 −45.52 44.07 17.08 30.58 44.11/43.55(0.99)

11792 D −4.91 −45.07 28.62 32.50 30.56 5.36/13.57(2.53)

11754 + TFA D −5.38 −53.48 34.09 25.85 29.97 5.58/7.55(1.35)

11681 + TFA F −4.75 −41.75 30.46 29.26 29.86 5.47/4.08(0.75)

11483 + TFA F −5.7 −45.23 29.55 28.99 29.27

12615 D −5.86 −44.34 31.59 26.88 29.24 5.12/5.93(1.16)

11617 D −6.05 −62.56 27.71 30.66 29.18 6.01/19.73(3.28)

11592 D −5.13 −65.33 24.41 33.91 29.16 21.82/18.28(0.84)

11725 + TFA D −5.93 −41.53 26.65 31.20 28.93 13.00/1.11(0.85)

11457 E −4.91 −34.84 38.67 18.48 28.58 27.40/48.97(1.79)

12623 + TFA D −5.2 −54.89 21.11 35.88 28.49 37.25/17.02(0.46)

12740 + TFA D −5.62 −56.42 28.77 27.91 28.34 49.36/23.63(0.48)

12684 + TFA D −5.56 −49.03 40.81 15.75 28.28 25.72/19.52(0.76)

12213 G −5.11 −42.88 28.35 27.66 28.01 39.11/38.60(0.99)

12227 G −4.6 −45.64 31.47 23.08 27.27 16.42/17.07(1.04)

11680 + TFA F −4.78 −38.29 32.59 21.89 27.24 21.86/17.38(0.80)

11609 D −6.98 −54.01 28.83 25.63 27.23 35.98/24.31(0.68)

12614 + TFA D −5.79 −46.31 20.34 34.09 27.21 19.42/7.56(0.39)

11663 + TFA G −6.6 −52.02 31.73 21.73 26.73 non-convergent

11816 + TFA E −4.77 −41.3 39.76 13.58 26.67 14.15/28.12(1.99)

12622 D −5.8 −48.37 21.30 31.34 26.32 20.80/11.52(0.55)

11624 D −5.58 −47.58 22.54 29.61 26.08 14.04/22.37(1.59)

12630 + TFA D −5.66 −41.51 17.06 34.97 26.02 11.70/8.75(0.75)

12229 + TFA E −3.71 −32.18 16.57 35.37 25.97 19.23/20.86(1.08)

12629 + TFA D −5.13 −42.48 19.83 31.95 25.89 12.66/10.71(0.85)

11777 G −3.31 −42.7 37.92 13.81 25.87 6.60/10.66(1.62)

11679 + TFA G −5.35 −46.98 31.02 20.64 25.83 12.55/8.73(0.70)

12676 E −3.44 −42.61 28.01 23.61 25.81 12.93/18.79(1.45)

11746 + TFA D −5.48 −45.63 29.51 21.86 25.68 33.53/14.36(0.43)

12743 + TFA D −4.99 −46.31 9.17 42.12 25.65 6.88/9.86(1.43)

11300 + TFA D −7.97 −65.69 25.11 26.12 25.61 26.34/7.95(0.30)

11751 D −6.76 −54.92 25.87 25.14 25.51 9.88/6.31(0.64)

12669 + TFA D −5.44 −42.18 11.73 39.10 25.42 49.32/36.71(0.74)

11807 + TFA G −5.01 −42.97 30.73 20.02 25.37 5.31/17.78(3.35)

11673 + TFA G −5.6 −42.41 33.30 17.39 25.35 non-convergent

12776 + TFA D −5.61 −43.06 21.34 29.30 25.32 29.74/19.03(0.64)

11670 + TFA G −5.37 −39.67 34.91 15.63 25.27 13.52/24.32(1.80)

11469 D −5.08 −60.97 26.79 23.71 25.25 10.12/11.47(1.13)

11625 + TFA D −5.97 −44.48 31.51 18.94 25.22 49.03/34.70(0.71)

12662 D −6.33 −40.08 21.64 28.79 25.21

12644 + TFA D −5.99 −43.95 16.45 33.67 25.06 46.89/30.86(0.66)

11674 + TFA G −4.7 −38.24 31.91 18.16 25.04 20.20/35.53(1.76)

Two-dimensional representations of the docked pose of AD4-1734, and AD4-10381 compounds, along with AD4-1734-like, AD4-10381-like compounds were produced.

Docking of compound AD4-1734 to EGFR is depicted, for example, in FIG. 8A. Docking of compound AD4-10631 (an AD4-1734-like compound) to EGFR is depicted, for example, in FIG. 8B. Docking of compound AD4-10188 (an AD4-1734-like compound) to EGFR is depicted, for example, in FIG. 8C. Docking of compound AD4-10186 (an AD4-1734-like compound) to EGFR is depicted, for example, in FIG. 8D. Docking of compound AD4-10633 (an AD4-1734-like compound) to EGFR is depicted, for example, in FIG. 8E. Docking of compound AD4-10174 (an AD4-1734-like compound) to EGFR is depicted, for example, in FIG. 8F.

Docking of compound AD4-10381 to EGFR is depicted, for example, in FIG. 9A. Docking of compound AD4-11340 (an AD4-10381-like compound) to EGFR is depicted, for example, in FIG. 9B. Docking of compound AD4-12632 (an AD4-10381-like compound) to EGFR is depicted, for example, in FIG. 9C. Docking of compound AD4-12681 (an AD4-10381-like compound) to EGFR is depicted, for example, in FIG. 9D. Docking of compound AD4-12732 (an AD4-10381-like compound) to EGFR is depicted, for example, in FIG. 9E. Docking of compound AD4-11511 (an AD4-10381-like compound) to EGFR is depicted, for example, in FIG. 9F.

Example 8 Combinatorial Studies

Studies were initiated to evaluate the ability of various compounds disclosed herein to synergize with several compounds known to inhibit the function of the EGF receptor in a cell proliferation assay (MTT assay). These compounds include Tarceva, Tykerb (non-selective inhibitor of EGFR and HER2 tyrosine kinases), Iressa (selective inhibitor of EGFR kinase), and a mouse antibody homolog of Erbitux (clone 225; inhibits binding of EGF to the EGF receptor). The rationale for this hypothesis is based on the idea that the AD4 compounds interact with a different site than the EGFR antibody, Erbitux, and have a different mechanism of action than the EGFR kinase inhibitors, Tykerb, Iressa or Tarceva.

Methods are according to Examples 1-7 except as indicated otherwise.

Known EGFR inhibitors Tykerb (AD4-0003), Iressa (AD4-0004), Tarceva (AD4-0005) and Clone 225 (from which Erbitux was derived) were titrated in the absence or presence of a fixed concentration of AD4 compound. The EGFR kinase inhibitors and AD4 compound were pre-diluted in 100% DMSO (DMSO+0.2% TFA for AD4 10381) as necessary such that a 1/200 dilution into DMEM+BSA resulted in 2× the final concentration desired. Clone 225 was diluted similarly with the exception that the pre-dilution was made in DMEM+BSA rather than DMSO. The EGFR inhibitor and AD4 compound dilutions were then mixed 1:1 in a 96 well plate. 50 μl of the mix was then added to the cell plate.

For the Clone 225 Combination Experiment, Clone 225 (Lab Vision/Thermo Scientific; #MS-269) was tested at concentration of 1, 0.5, 0.25, 0.125, 0.0625 and 0 μg/ml. The EGF concentration used for stimulation was 10 ng/ml. For the Tarceva Combination Experiment, Tarceva was tested at concentrations of 156, 63, 25, 10, 4 and 0 nM. The EGF concentration used for stimulation was 5 ng/ml. For the Tykerb Combination Experiment, Tykerb was tested at concentrations of 78, 31.25, 12.5, 5, 2 and 0 nM. The EGF concentration used for stimulation was 5 ng/ml. For the Iressa Combination Experiment, Iressa was tested at concentrations of 156, 63, 25, 10, 4 and 0 nM. The EGF concentration used for stimulation was 5 ng/ml.

The concentration of AD4 compound used in each experiment are provided in the graphs and data tables. A shifting of the inhibitor curve to the left indicates an increase in the effectiveness of the AD4-compound.

In these studies, the ability of an AD4 compound and the known compound (e.g. Tykerb), either alone or combined in a fixed constant ratio, were evaluated for their ability to inhibit cell proliferation in the MTT assay. From these studies, the following values were calculated: IC₅₀ values for the AD4 compound alone, for Tykerb (or other test compound) alone, and for each compound when combined; the Combination Index (CI), which reflects the degree of antagonism or synergism (see TABLE 13 below); and the Dose Reduction Index (DRI), which is a measure of how many fold the dose of each drug in a synergistic combination may be reduced at a given effect level when compared with the doses of each drug alone.

TABLE 13 Combination Index KEY: Range of CI Description Graded Symbols   <0.1 Very strong synergism + + + + + 0.1-0.3 Strong synergism + + + + 0.3-0.7 Synergism + + +  0.7-0.85 Moderate synergism + + 0.85-0.9  Slight synergism +  0.9-1.10 Nearly additive +/− 1.10-1.20 Slight antagonism − 1.20-1.45 Moderate antagonism − − 1.45-3.3  Antagonism − − − 3.3-10  Strong antagonism − − − − >10 Very strong antagonism − − − − −

Results showed the following. A series of AD4-Pharma compounds produce a synergistic effect with Tykerb, Iressa and Erbitux to enhance their effect in a cell proliferation assay. These synergistic effects were demonstrated by significant changes in the Dose Reduction Index, the Combination Index and shifts in the dose-response curves.

The effects of a number of compounds appear to involve positive cooperativity because the effect increases as the concentration of the compound increases. Compounds that demonstrated the greatest degree of positive co-operativity usually demonstrated high DRI values. Some of the compounds that demonstrate the greatest degree of positive co-operativity also show synergistic behavior as evidenced by a low CI value. Iressa and Tarceva, another selective inhibitor of EGFR kinase, did not produce synergistic effects with Tykerb. Compounds acting at the same target (i.e., EGFR kinase) should not be synergistic.

An example of a shift in the dose-response curve is shown in FIG. 10A-FIG. 10B, where AD4-10628 produced a leftward shift (higher potency) in the dose-response curves for both Tykerb and Iressa. The effect is more evident at higher concentrations of the compound, indicating a positive co-operativity effect may be involved in the compound's action.

Results for several of the more potent compounds are summarized in TABLE 14. AD4-10628 AD4-11511 produce a leftward shift (i.e., greater potency) in Tykerb's dose-response curve, and demonstrate very high DRI values. Although significant shifts in the dose-response curves for the EGFR kinase inhibitors are produced, these effects are not translated into significant shifts in the IC₅₀ values (50% inhibition), since the effects of the AD4 compounds are observed to a greater degree at higher concentrations.

TABLE 14 Summary of Median Effect Analysis Studies IC₅₀ Tykerb IC₅₀ Tykerb + DRI @ CI @ Compound (nM) Compound (nM) ED97 ED90 AD4-10628 178 59 126 0.55 AD4-11511 189 118 84 0.87 Iressa 307 160 2 1.15 IC₅₀ Iressa IC₅₀ Iressa + DRI @ CI @ (nM) Compound (nM) ED97 ED90 AD4-10628 207 93 105 0.91 AD4-11511 326 245 96 0.93

DRI values were calculated for the effect of the test compound on Tykerb, as well as for Tykerb's effect on the test compound. In general, most compounds enhanced the effect of Tykerb, as demonstrated by a high DRI, whereas Tykerb usually had a minimal effect on the test compound. As a result, the reported DRI is for the effect of the test compound on Tykerb. This can best be observed by plotting DRI as a function of Fa, or percent effect, ranging from 5% to 97% (see e.g., FIG. 11). AD4-10628 had a significant effect on the activity of Tykerb, which becomes greater at higher activity levels (or doses), whereas Tykerb has little effect on AD4-10628 (see e.g., FIG. 11). As shown in TABLE 14, AD4-10628 and AD4-11511 all produced a fairly high DRI for both Tykerb and Iressa.

CI values were calculated where the combined effect of the compounds produces 50%, 90%, 95% and 97% (i.e., ED₅₀, ED₉₀, ED₉₅ and ED₉₇) inhibition of cell proliferation. A graph showing the CI values at 90% inhibition is displayed in FIG. 12. Response below the red line (i.e CI<0.9) indicates synergism. As indicated in FIG. 12, a number of compounds demonstrated synergism. For example, the CI values for AD4-10628 (see TABLE 14) demonstrated significant synergy with Tykerb. In contrast, only AD4-1505 shows synergy with Iressa based on the CI value (data not shown). Since Iressa and Tykerb have similar mechanisms of action, minimal interaction would be expected between these two compounds. As shown in TABLE 14, little interaction is observed based on the DRI and CI values for Iressa.

These results show that the AD4 compounds produce a significant effect on EGF receptor-mediated cell proliferation through a site that is distinct from either EGF receptor kinase or the EGF receptor. Furthermore, based on their synergistic effect, the compounds may provide a unique method to achieve the same or enhanced therapeutic effect while using a lower therapeutic dose of the marketed compounds, Tykerb, Iressa or Erbitux.

Example 9 Synthesis of Compounds

The following example describes synthesis of AD4-12632, which is an AD4-11511-like compound.

Step 1 was as follows (see e.g., Hamann 1988 J. Med. Chem. 41(4), 623-639):

m-Toluidine (across, 32.1 g, 0.3 mol) was dissolved in 250 ml of acetone and treated with 100 g of MgSO4, iodine (2.6 g, 0.01 mol) and a catalytic amount (200 mg) of t-butyl catechol. The mixture was stirred and heated to reflux temperature overnight. After cooling to room temperature the dark mixture was filtered through Celite which was rinsed with EtOAc. The solution was treated with decolorizing charcoal, filtered, and concentrated using a rotovap. The dark residual oil was purified by fractional distillation. The fraction boiling between 90-95° C. @ 0.05 mm/Hg contained the desired product. The light yellow oil solidified after cooling in the refrigerator to give the free base as an off-white waxy solid. The free base was converted to the hydrochloride salt as follows. The freebase was dissolved in Et₂O and cooled in an ice bath. A solution of hydrogen chloride in Et₂O was prepared and a slight molar excess (1.1 equivalents) was added from a dropping funnel. The mixture was stirred at 0° C. for 1 h and then filtered to collect 1,2-dihydro-2,2,4,7-tetramethylquinoline hydrochloride as a white solid (MP 213-215° C.).

Step 2 was as follows (see e.g., Web 2003 Bioorg. Med. Chem. 11, 77-85):

1,2-dihydro-2,2,4,7-tetramethylquinoline hydrochloride (9.35 g, 0.004 mol) was suspended with stirring in 30 ml water and 20 ml of EtOH was added followed by 2-cyanoguanidine (Acros, 3.4 g, 0.004 mol). The heterogeneous mixture was heated to 100° C. for 6 h and then cooled to room temperature. The solid was collected by filtration and washed with 500 ml of cold water followed by 100 ml of 2-propanol. The off-white solid was dried and then dissolved by heating to 150° C. in DMSO. Upon cooling to room temperature a white solid formed and was collected by filtration. The solid was rinsed with Et2O and dried to give 4,7-dimethylquinazoyl-2-guanidine hydrochloride as a white solid (MP 329-331° C.). The hydrochloride salt was suspended in 5N aqueous sodium hydroxide and stirred at room temperature for 5 days. The white solid was collected by filtration and rinsed with 2×250 ml water and 100 ml of ice-cold 2-propanol. The solid was dried to give 4,7-dimethylquinazoyl-2-guanidine free base as an off white solid (MP 260-261° C.).

Step 3 was as follows (see e.g., Shikhaliev 2002 Chem. Het. Compounds 38(11), 1368-1370):

4,7-dimethylquinazoyl-2-guanidine (2.15 g, 0.01 mol) and ethyl-2-methyl acetoacetate (1.58 g, 0.011 mol) were stirred in 15 ml DMSO to produce a suspension. The solution was heated to 130° C. overnight to give a clear brown solution and then cooled to room temperature. The solid that formed was collected by filtration. The solid was dissolved in EtOH at room temperature with stirring, treated with decolorizing charcoal and filtered through a bed of Celite. The EtOH solution was cooled to 0° C. to give an off white solid that is collected by filtration (MP 226-228° C.). The resulting product was the AD4-12632 compound. 

What is claimed is:
 1. A compound having a formula of:

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein, R¹⁰¹ is selected from the group consisting of: (i) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (ii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (iii) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of: (a) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (b) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (c) heteroaryl containing from 1 to 4 N, O, or S atoms; R¹⁰² is hydrogen; R¹⁰³ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (v) halogen; R¹⁰⁴ and R¹⁰⁵ are independently selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (iv) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; R¹⁰⁶ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of: (a) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (b) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (c) heteroaryl containing from 1 to 4 N, O, or S atoms; (d) aryl comprising a phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; (e) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (f) trifluoromethyl; (g) trifluoromethoxy; (h) difluoromethoxy; (i) 3,4-methylenedioxy; (j) 2,3-methylenedioxy; (k) nitro; and (l) halogen; and (iv) C-1 to C-3 substituted alkyl having at least one substitution group selected from the group consisting of: (a) carboxyl —COOR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of:  (1) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation;  (2) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (3) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;  (4) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (A) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (5) trifluoromethyl;  (6) trifluoromethoxy;  (7) difluoromethoxy;  (8) 3,4-methylenedioxy;  (9) 2,3-methylenedioxy;  (10) nitro; and  (11) halogen; and (c) aryloxy (—OAr); R¹⁰⁷ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of: (a) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (b) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (c) aryl comprising a phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; (d) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (e) trifluoromethyl; (f) trifluoromethoxy; (g) difluoromethoxy; (h) 3,4-methylenedioxy; (i) 2,3-methylenedioxy; (j) nitro; and (k) halogen; and (v) C-1 to C-3 substituted alkyl having at least one substitution group selected from the group consisting of: (a) carboxyl —COOR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of:  (1) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation;  (2) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (3) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;  (4) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (A) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (5) trifluoromethyl;  (6) trifluoromethoxy;  (7) difluoromethoxy;  (8) 3,4-methylenedioxy;  (9) 2,3-methylenedioxy;  (10) nitro; and  (11) halogen; and (c) aryloxy (—OAr); and Formula (25) excludes a compound of Formula (24):

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein, R¹⁰⁸ is selected from the group consisting of: (i) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (ii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iii) C-1 to C-3 substituted alkyl having at least one substitution group selected from the group consisting of: (a) carboxyl —COOR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of:  (1) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation;  (2) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (3) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;  (4) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (A) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (5) trifluoromethyl;  (6) trifluoromethoxy;  (7) difluoromethoxy;  (8) 3,4-methylenedioxy;  (9) 2,3-methylenedioxy;  (10) nitro; and  (11) halogen; and (c) aryloxy (—OAr); (iv) aryl comprising a phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; (v) amino; and (vi) hydroxyl; R¹⁰⁹ is hydrogen; R¹¹⁰ and R¹¹¹ are independently selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) C-1 to C-3 substituted alkyl having at least one substitution group selected from the group consisting of: (a) carboxyl —COOR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of:  (1) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation;  (2) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (3) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;  (4) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (A) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (5) trifluoromethyl;  (6) trifluoromethoxy;  (7) difluoromethoxy;  (8) 3,4-methylenedioxy;  (9) 2,3-methylenedioxy;  (10) nitro; and  (11) halogen; and (c) aryloxy (—OAr); (v) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (vi) halogen; R¹¹² is selected from the group consisting of: hydrogen and straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R¹¹³ is selected from the group consisting of: (i) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (ii) trifluoromethyl; (iii) aryl comprising a phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; and (iv) amino; R¹¹⁴ is selected from the group consisting of: (i) hydrogen; (ii) acyl —COR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (iii) carboxyl —COOR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; R¹¹⁵ is selected from the group consisting of: (i) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (ii) trifluoromethyl; and (iii) aryl comprising a phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; and Formula (27) excludes a compound of Formula (24):

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein n is 1 or 2; R¹¹⁶ is selected from the group consisting of: (i) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (ii) aryl comprising a phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; R¹¹⁷ of is hydrogen; R¹¹⁸ is selected from the group consisting of: (i) hydrogen, (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (ii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (iii) halogen; R¹¹⁹ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (iii) aryl comprising a phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; R¹²⁰ is selected from the group consisting of: (i) hydrogen, (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (iii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; R¹²¹ is selected from the group consisting of: hydrogen and aryl comprising a phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; and Formula (28) excludes a compound of Formula (24):


2. The compound of claim 1, wherein (A) the compound is according to Formula (25) (10381-like Type D) and at least one of the following features is satisfied R¹⁰¹ is selected from the group consisting of methyl and phenyl; R¹⁰³ is selected from the group consisting of methyl, ethyl, chloro, methoxy, and ethoxy; R¹⁰⁴ is selected from the group consisting of hydrogen, methyl, and methoxy; R¹⁰⁵ is selected from the group consisting of hydrogen, methyl, methoxy, and ethoxy; R¹⁰⁶ is selected from the group consisting of methyl and phenyl; or the compound is selected from the group consisting of

(B) the compound is according to Formula (27) (10381-like Type E) and at least one of the following features is satisfied R¹⁰⁸ is selected from the group consisting of: methyl, phenyl, and hydroxyl; R¹¹⁰ and R¹¹¹ are independently selected from the group consisting of: hydrogen, methyl, methoxy, and chloro; R¹¹² is methyl; R¹¹³ is selected from the group consisting of: methyl, phenyl, and amino; R¹¹⁵ is selected from the group consisting of: methyl, phenyl, and trifluoromethyl; the compound is selected from the group consisting of

or (C) the compound is according to Formula (28) (10381-like Type F) and at least one of the following features is satisfied n is 2 and the compound has a formula of:

or the compound is


3. The compound of claim 1 that inhibits EGFR activity comprising: eight or more of functional groups F(IV)1, F(IV)2, F(IV)3, F(IV)4, F(IV)5, F(IV)6, F(IV)7, F(IV)8, F(IV)9, and F(IV)10 of a Scheme IV 10381-like pharmacophore; wherein functional group F(IV)1 accepts a hydrogen bond from receptor side chain OH of Thr239 of SEQ ID NO: 1 and has coordinates of r=49.686, θ (theta)=113.993, and φ (phi)=−17.014 and a spherical radius of about 1.2 Å; functional group F(IV)2 accepts a hydrogen bond from receptor side chain OH of Thr239 of SEQ ID NO: 1 and has coordinates of r=48.071, θ (theta)=115.388, and φ (phi)=−16.211 and a spherical radius of about 1.6 Å; functional group F(IV)3 forms a hydrophobic contact with a side chain of receptor Met576 and imidazolidine ring of Pro242 of SEQ ID NO: 1 and has coordinates of r=50.781, θ (theta)=113.121, and φ (phi)=−17.520 and a spherical radius of about 1.2 Å; functional group F(IV)4 forms a hydrophobic contact with a side chain of receptor Met576 and imidazolidine ring of Pro242 of SEQ ID NO: 1 and has coordinates of r=52.021, θ (theta)=114.264, and φ (phi)=−15.878 and a spherical radius of about 1.5 Å; functional group F(IV)5 forms a hydrophobic contact with a side chain of receptor Met576 and imidazolidine ring of Pro242 of SEQ ID NO: 1 and has coordinates of r=50.322, θ (theta)=111.253, and φ (phi)=−15.426 and a spherical radius of about 1.2 Å; functional group F(IV)6 forms a hydrophobic contact with a side chain of receptor Met576 and imidazolidine ring of Pro242 of SEQ ID NO: 1 and has coordinates of r=51.575, θ (theta)=112.433, and φ (phi)=−13.827 and a spherical radius of about 1.5 Å; functional group F(IV)7 forms a hydrophobic contact with a side chain of receptor Leu243 and a side chain of Thr239 of SEQ ID NO: 1 and has coordinates of r=47.767, θ (theta)=112.521, and φ (phi)=−10.196 and a spherical radius of about 1.2 Å; functional group F(IV)8 donates a hydrogen bond to a backbone carbonyl of His280 of SEQ ID NO: 1 and has coordinates of r=45.184, θ (theta)=112.328, and φ (phi)=−8.875 and a spherical radius of about 1.2 Å; functional group F(IV)9 forms a hydrophobic contact with a side chain of receptor Met244 and Leu243 of SEQ ID NO: 1 and has coordinates of r=49.512, θ (theta)=108.007, and φ (phi)=−8.504 and a spherical radius of about 1.2 Å; functional group F(IV)10 forms a hydrophobic contact with a side chain of receptor Met244 of SEQ ID NO: 1 and has coordinates of r=50.431, θ (theta)=104.985, and φ (phi)=−7.420 and a spherical radius of about 1.8 Å; and the compound substantially maintains a non-extended tether inactive configuration of EGFR or substantially prevents stabilization of an extended tether active configuration of EGFR.
 4. A compound having a formula of:

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein, R²³ and R²⁷ are hydrogen; R²⁴ and R²⁶ are independently selected from the group consisting of: (i) hydrogen; (ii) C-1 to C-6 lower alkyl; (iii) alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (iv) halogen; R²⁵ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) C-1 to C-3 substituted alkyl having at least one substitution group selected from the group consisting of (a) carboxyl —COOR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of: straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; trifluoromethyl; trifluoromethoxy; difluoromethoxy; 3,4-methylenedioxy; 2,3-methylenedioxy; nitro; and halogen; and (c) aryloxy (—OAr); (v) alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (vi) halogen; R²⁸ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (iii) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of: (a) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (b) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (c) halogen. R²⁹ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) hydroxy; (iv) alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (v) halogen; R³⁰ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) trifluoromethyl; and (v) halogen; R³¹ is selected from the group consisting of: (i) hydrogen; (ii) hydroxy; (iii) alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (v) trifluoromethyl; and (vi) halogen; and R³² is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) hydroxy; (iv) trifluoromethyl; and (v) halogen; and Formula (13) excludes the compound of Formula (12):

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein, R³³ is selected from the group consisting of: (i) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (ii) C-1 to C-3 substituted alkyl having at least one substitution group selected from the group consisting of: (a) carboxyl —COOR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of: straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; trifluoromethyl; trifluoromethoxy; difluoromethoxy; 3,4-methylenedioxy; 2,3-methylenedioxy; nitro; and halogen; and (c) aryloxy (—OAr); and (iii) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of: (a) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (b) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (c) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; (d) alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (e) trifluoromethyl; (f) trifluoromethoxy; (g) difluoromethoxy; (h) 3,4-methylenedioxy; (i) 2,3-methylenedioxy; (j) nitro; and (k) halogen; R³⁴ is hydrogen; R³⁵ is selected from the group consisting of: (i) hydrogen; (ii) hydroxy; (iii) alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (v) trifluoromethyl; and (vi) halogen; R³⁶ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) hydroxy; (iv) alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (v) trifluoromethyl; and (vi) halogen; and R³⁷ is hydrogen; and Formula (14) excludes a compound of Formula (12):

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein, n is 1 or 2; X² and X³ are independently selected from the group consisting of: carbon, oxygen, nitrogen, and sulfur; R³⁸ is hydrogen; R³⁹ is selected from the group consisting of: (i) hydrogen; (ii) C-1 to C-6 lower alkyl; (iii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (iv) halogen; R⁴⁰ is hydrogen; R⁴¹ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (iii) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of: (a) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (b) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (c) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; (d) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (e) trifluoromethyl; (f) trifluoromethoxy; (g) difluoromethoxy; (h) 3,4-methylenedioxy; (i) 2,3-methylenedioxy; (j) nitro; and (k) halogen; R⁴² is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) hydroxy; (iv) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (v) halogen; R⁴³ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) trifluoromethyl; and (v) halogen; R⁴⁴ is selected from the group consisting of: (i) hydrogen; (ii) hydroxy; (iii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (v) trifluoromethyl; and (vi) halogen; R⁴⁵ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) hydroxy; (iv) trifluoromethyl; and (v) halogen; and Formula (15) excludes the compound of Formula (12):

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein, n is 1 or 2; X⁴ and X⁵ are independently selected from the group consisting of: carbon, oxygen, nitrogen and sulfur; R⁴⁶ and R⁵⁰ are hydrogen; R⁴⁷ and R⁴⁹ are independently selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) halogen; and (v) R⁴⁷ and R⁴⁹ form a 5 or 6 membered cycloalkyl ring; R⁴⁸ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) C-1 to C-3 substituted alkyl having at least one substitution group selected from the group consisting of: (a) carboxyl —COOR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of:  (1) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation;  (2) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (3) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;  (4) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (A) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (5) trifluoromethyl;  (6) trifluoromethoxy;  (7) difluoromethoxy;  (8) 3,4-methylenedioxy;  (9) 2,3-methylenedioxy;  (10) nitro; and  (11) halogen; and (c) aryloxy (—OAr); (v) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (vi) halogen; and (vii) R⁴⁸ forms a 5 or 6 membered cycloalkyl ring with R⁴⁷; R⁵¹ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (iii) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of: (a) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (b) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (c) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; (d) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (e) trifluoromethyl; (f) trifluoromethoxy; (g) difluoromethoxy; (h) 3,4-methylenedioxy; (i) 2,3-methylenedioxy; (j) nitro; and (k) halogen; R⁵² is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) hydroxy; (iv) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (v) halogen; R⁵³ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) hydroxy; (iv) trifluoromethyl; and (v) halogen; and Formula (16) excludes the compound of Formula (12):


5. The compound of claim 4, wherein (A) the compound is according to Formula (13) (1734-like Type P) and one or more of the following features are satisfied: R²⁴ and R²⁶ are independently selected from the group consisting of: hydrogen and alkoxy; R²⁵ is selected from the group consisting of: hydrogen or alkoxy —OR¹⁰; R²⁸ is selected from the group consisting of: hydrogen or aryl; R²⁸ is hydrogen; R³⁰ is straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R³¹ is selected from the group consisting of: hydroxy or alkoxy —OR¹⁰; R³² is hydrogen; or the compound is selected from the group consisting of

(B) the compound is according to Formula (14) (1734-like Type Q) and one or more of the following features are satisfied: R³⁵ is straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R³⁶ is selected from the group consisting of: (i) hydroxy and (ii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; or the compound is

(C) the compound is according to Formula (15) (1734-like Type R) and one or more of the following features are satisfied: n is 1; X² and X³ are independently selected from the group consisting of: oxygen or carbon; R⁴² is hydrogen; R⁴³ is straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R⁴⁴ is selected from the group consisting of (i) hydroxy and (ii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; R⁴⁵ is hydrogen; or the compound is

or (D) the compound is according to Formula (16) (1734-like Type S) and one or more of the following features are satisfied: n is 1; X⁴ and X⁵ are independently selected from the group consisting of oxygen and carbon; R⁴⁸ is selected from the group consisting of hydrogen or straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R⁴⁷ and R⁴⁸ form a 5 membered cycloalkyl ring; R⁵¹ is selected from the group consisting of (i) hydrogen and (ii) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of: (a) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation, (b) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom, (c) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms, (d) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation, and (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom, (e) trifluoromethyl, (f) trifluoromethoxy, (g) difluoromethoxy, (h) 3,4-methylenedioxy, (i) 2,3-methylenedioxy, (j) nitro, and (k) halogen; R⁵² is hydrogen; or the compound is selected from the group consisting of


6. The compound of claim 4 that inhibits EGFR activity, the compound comprising: five or more of functional groups F(II)1, F(II)2, F(II)3, F(II)4, F(II)5, F(II)6, F(II)7, F(II)8, and F(II)9 of a Scheme II 1734-like pharmacophore; wherein functional group F(II)1 donates an H-bond or forms a salt bridge to a carboxylate side chain of receptor Asp553 of SEQ ID NO: 1 and has coordinates of r=56.363, θ (theta)=94.368, and φ (phi)=−17.752 and a spherical radius of about 1.2 Å; functional group F(II)2 is a donor and has coordinates of r=53.290, θ (theta)=101.494, and φ (phi)=−23.244 and a spherical radius of about 1.0 Å; functional group F(II)3 forms a hydrophobic contact with a side chain of receptor Val568, imidazolidine side chain of receptor Pro552 and with a side chain of Met253 of SEQ ID NO: 1 and has coordinates of r=53.726, θ (theta)=97.830, and φ (phi)=−18.377 and a spherical radius of about 1.7 Å; functional group F(II)4 forms a hydrophobic contact with side chain of receptor Val575, Met253, and with an imidazolidine ring of receptor Pro552 of SEQ ID NO: 1 and has coordinates of r=53.647, θ (theta)=103.844, and φ (phi)=−20.990 and a spherical radius of about 1.4 Å; functional group F(II)5 donates an H-bond to a side chain hydroxyl of Thr570 of SEQ ID NO: 1 and has coordinates of r=51.093, θ (theta)=104.261, and φ (phi)=−25.552 and a spherical radius of about 1.2 Å; functional group F(II)6 is a donor having directionality of F4 with respect to a backbone carbonyl of receptor Thr570 of SEQ ID NO: 1 and has coordinates of r=52.340, θ (theta)=103.980, and φ (phi)=−27.461 and a spherical radius of about 1.5 Å; functional group F(II)7 accepts an H-bond from a receptor backbone NH of Ala573 of SEQ ID NO: 1 and has coordinates of r=51.383, θ (theta)=106.455, and φ (phi)=−24.319 and a spherical radius of about 1.2 Å; functional group F(II)8 is an acceptor having directionality of F7 with respect to the backbone NH of receptor Ala573 of SEQ ID NO: 1 and has coordinates of r=52.861, θ (theta)=107.691, and φ (phi)=−25.448 and a spherical radius of about 1.5 Å; functional group F(II)9 donates an H-bond to a backbone carbonyl of Asp563 and forms a salt bridge to a side chain carboxylate of receptor Asp563 of SEQ ID NO: 1 and has coordinates of r=57.688, θ (theta)=99.198, and φ (phi)=−21.588 and a spherical radius of about 1.2 Å; and the compound substantially maintains a non-extended tether inactive configuration of EGFR or substantially prevents stabilization of an extended tether active configuration of EGFR.
 7. A compound having a formula of:

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein, R⁵⁴ is selected from the group consisting of: (i) a 5 or 6 membered heterocyclic containing from 1 to 4 N, O, or S atoms or any combination of those atoms with carbon atoms to form a heterocyclic aromatic ring optionally substituted with from 1 to 3 groups selected from: (a) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (b) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (c) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (d) trifluoromethyl, (e) trifluoromethoxy, (f) substituted amino, (g) nitro; and (h) halogen; R⁵⁵ is selected from the group consisting of: hydrogen and straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R⁵⁶, R⁵⁷, R⁵⁸, and R⁵⁹ are hydrogen; R⁶⁰ is selected from the group consisting of: (i) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (ii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (iii) C-1 to C-3 substituted alkyl having at least one substitution group selected from the group consisting of: (a) carboxyl —COOR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of:  (1) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation;  (2) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (3) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;  (4) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (A) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (5) trifluoromethyl;  (6) trifluoromethoxy;  (7) difluoromethoxy;  (8) 3,4-methylenedioxy;  (9) 2,3-methylenedioxy;  (10) nitro; and  (11) halogen; and (c) aryloxy (—OAr); and Formula (18) excludes a compound of Formula (17):

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein, R⁶¹, R⁶², and R⁶⁴ are independently selected from the group consisting of: hydrogen and straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R⁶³ and R⁶⁵ are independently selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (iii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; R⁶⁶ is selected from the group consisting of: hydrogen and straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R⁶⁷, R⁶⁸, R⁶⁹, and R⁷⁰ are hydrogen; R⁷¹ is selected from the group consisting of: (i) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (ii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iii) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of: (a) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (b) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (c) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; (d) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (e) trifluoromethyl; (f) trifluoromethoxy; (g) difluoromethoxy; (h) 3,4-methylenedioxy; (i) 2,3-methylenedioxy; (j) nitro; and (k) halogen; (iv) C-1 to C-3 substituted alkyl having at least one substitution group selected from the group consisting of: (a) carboxyl —COOR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of:  (1) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation;  (2) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (3) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;  (4) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (A) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (5) trifluoromethyl;  (6) trifluoromethoxy;  (7) difluoromethoxy;  (8) 3,4-methylenedioxy;  (9) 2,3-methylenedioxy;  (10) nitro; and  (11) halogen; and (c) aryloxy (—OAr); (v) heteroaryl containing a heterocyclic ring containing from 1 to 4 N, O, or S atoms optionally substituted with one or more groups selected from the group consisting of: (a) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (b) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (c) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; (d) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (e) trifluoromethyl; (f) trifluoromethoxy; (g) nitro; and (h) halogen; and Formula (19) excludes a compound of Formula (17):

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein, R⁷² is selected from the group consisting of: hydrogen and straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R⁷³ is selected from the group consisting of: (i) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (ii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iii) C-1 to C-3 substituted alkyl having at least one substitution group selected from the group consisting of: (a) carboxyl —COOR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of:  (1) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation;  (2) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (3) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;  (4) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (A) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (5) trifluoromethyl;  (6) trifluoromethoxy;  (7) difluoromethoxy;  (8) 3,4-methylenedioxy;  (9) 2,3-methylenedioxy;  (10) nitro; and  (11) halogen; and (c) aryloxy (—OAr); and (iv) acyl —COR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; R⁷⁴, R⁷⁵, R⁷⁶, and R⁷⁷ are hydrogen; and R⁷⁸ is selected from the group consisting of: (i) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (ii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iii) C-1 to C-3 substituted alkyl having at least one substitution group selected from the group consisting of: (a) carboxyl —COOR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of:  (1) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation;  (2) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (3) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;  (4) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (A) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation;  (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (5) trifluoromethyl;  (6) trifluoromethoxy;  (7) difluoromethoxy;  (8) 3,4-methylenedioxy;  (9) 2,3-methylenedioxy;  (10) nitro; and  (11) halogen; and (c) aryloxy (—OAr); and Formula (20) excludes a compound of Formula (17):

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein, n is 1 or 2; R⁷⁹, R⁸¹, R⁸², R⁸³, R⁸⁴, R⁸⁵, and R⁸⁶ are hydrogen; R⁸⁰ is selected from the group consisting of: (i) hydrogen, (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) alkoxy —OR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) halogen; and (v) trifluoromethyl; R⁸⁷ is selected from the group consisting of: (i) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (ii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iii) C-1 to C-3 substituted alkyl having at least one substitution group selected from the group consisting of: (a) carboxyl —COOR¹⁰ where R¹⁰ is selected from the group consisting of:  (1) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (2) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of:  (1) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; \  (2) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (3) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;  (4) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (A) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (5) trifluoromethyl;  (6) trifluoromethoxy;  (7) difluoromethoxy;  (8) 3,4-methylenedioxy;  (9) 2,3-methylenedioxy;  (10) nitro; and  (11) halogen; and (c) aryloxy (—OAr); and (iii) aryl comprising a phenyl or heteroaryl five or six membered ring containing from 1 to 4 N, O, or S atoms; and Formula (21) excludes a compound of Formula (17):

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein, n is 3, 4, or 5; R⁸⁸ is selected from the group consisting of: hydrogen and straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R⁸⁹, R⁹⁰, R⁹¹, R⁸³, and R⁹² are hydrogen; R⁹³ is selected from the group consisting of: (i) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (ii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iii) C-1 to C-3 substituted alkyl having at least one substitution group selected from the group consisting of: (a) carboxyl —COOR¹⁰ where R¹⁰ is a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation or a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (b) aryl having an unsubstituted phenyl ring or a phenyl ring substituted at the 2-, 3-, 4-, 5- or 6-position with one or more groups selected from the group consisting of:  (1) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation;  (2) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (3) aryl having phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms;  (4) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of:  (A) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and  (B) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom;  (5) trifluoromethyl;  (6) trifluoromethoxy;  (7) difluoromethoxy;  (8) 3,4-methylenedioxy;  (9) 2,3-methylenedioxy;  (10) nitro; and  (11) halogen; and (c) aryloxy (—OAr); and (iv) aryl comprising a phenyl or heteroaryl five or six membered ring containing from 1 to 4 N, O, or S atoms; and Formula (22) excludes a compound of Formula (17):

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein, R⁹⁴ is a 5 or 6 membered heterocyclic aromatic ring containing from 1 to 4 N, O, or S atoms or any combination of those atoms with carbon atoms optionally substituted with from 1 to 3 of groups selected from: (i) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (ii) C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) trifluoromethyl; (v) trifluoromethoxy; (vi) substituted amino; (vii) nitro; and (viii) halogen; R⁹⁵ is selected from the group consisting of: hydrogen and straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R⁹⁶, R⁹⁷, R⁹⁸, and R⁹⁹ are hydrogen; R¹⁰⁰ is aryl comprising a phenyl or heteroaryl containing from 1 to 4 N, O, or S atoms; and Formula (23) excludes a compound of Formula (17):


8. The compound of claim 7, wherein (A) the compound is according to Formula (18) (1886-like Type J) and one or more of the following features are satisfied: R⁵⁴ is selected from the group consisting of: 4,6-dimethyl-2-pyrimidine; 2,6-dimethoxy-4-pyrimidine; 6-methoxy-4-pyrimidine; 5-ethyl-2-(1,3,4-Thiadiazole); 5-methyl-3-Isoxazole; 3-methoxy-6-pyridazinamine, 2-thiazole; and 2-methoxy-3-pyrazine. R⁵⁵ is selected from the group consisting of hydrogen and methyl; the compound is selected from the group consisting of:

(B) the compound according to Formula (19) (1886-like Type K) is

(C) the compound according to Formula (20) (1886-like Type L) is selected from the group consisting of:

(D) the compound according to Formula (21) (1886-like Type M) is

(E) the compound of Formula (22) (1886-like Type N) is selected from the group consisting of:

or (F) the compound of Formula (23) (1886-like Type O) is selected from the group consisting of:


9. The compound of claim 7 that inhibits EGFR activity comprising: at least seven or more of functional groups F(III)1, F(III)2, F(III)3, F(III)4, F(III)5, F(III)6, F(III)7, F(III)8, and F(III)9 of a Scheme III 1886-like pharmacophore; wherein functional group F(III)1 donates a hydrogen bond to a side chain carbonyl of receptor Gln408 and accepts a hydrogen bond from the side chain NH₂ of receptor Gln384 of SEQ ID NO: 1 and has coordinates of r=81.552, θ (theta)=41.243, and φ (phi)=45.369 and a spherical radius of about 1.2 Å; functional group F(III)2 accepts a hydrogen bond from a side chain OH of receptor Ser440 of SEQ ID NO: 1 and has coordinates of r=87.287, θ (theta)=40.739, and φ (phi)=51.781 and a spherical radius of about 1.2 Å; functional group F(III)3 accepts a hydrogen bond from a side chain OH of receptor Ser440 of SEQ ID NO: 1 and has coordinates of r=86.320, θ (theta)=41.915, and φ (phi)=52.323 and a spherical radius of about 1.5 Å; functional group F(III)4 forms a favorable coulombic interaction with an imidazole side chain of receptor His409 of SEQ ID NO: 1 and has coordinates of r=85.870, θ (theta)=38.463, and φ (phi)=41.650 and a spherical radius of about 1.2 Å; functional group F(III)5 forms a favorable coulombic interaction with an imidazole side chain of receptor His409 of SEQ ID NO: 1 and has coordinates of r=82.241, θ (theta)=37.431, and φ (phi)=44.151 and a spherical radius of about 1.5 Å; functional group F(III)6 accepts a hydrogen bond from, or forms a salt bridge to, NH³⁺ of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=88.009, θ (theta)=37.822, and φ (phi)=54.903 and a spherical radius of about 1.2 Å; functional group F(III)7 accepts a hydrogen bond from, or forms a salt bridge to, NH³⁺ of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=86.513, θ (theta)=36.889, and φ (phi)=54.484 and a spherical radius of about 1.5 Å; functional group F(III)8 donates a hydrogen bond to a side chain carbonyl of receptor Gln408 of SEQ ID NO: 1 and has coordinates of r=81.552, θ (theta)=41.243, and φ (phi)=45.369 and a spherical radius of about 1.2 Å; functional group F(III)9 donates a hydrogen bond to a side chain carbonyl of receptor Gln408 of SEQ ID NO: 1 and has coordinates of r=79.652, θ (theta)=40.928, and φ (phi)=44.528 and a spherical radius of about 1.6 Å; and the compound substantially maintains a non-extended tether inactive configuration of EGFR or substantially prevents stabilization of an extended tether active configuration of EGFR.
 10. A compound having a formula of:

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein, R¹²² and R¹²⁶ are independently selected from the group consisting of: (i) hydrogen, (ii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iii) trifluoromethyl; and (iv) halogen; R¹²³ and R¹²⁵ are independently selected from the group consisting of: (i) hydrogen; (ii) trifluoromethyl; (iii) halogen; (iv) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (v) acyl —COR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (vi) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; R¹²⁴ is selected from the group consisting of: (i) hydrogen; (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; (iii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; (iv) acyl —COR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (v) halogen; R¹²⁷ and R¹³¹ are independently selected from the group consisting of: (i) hydrogen, (ii) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (iii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; R¹²⁸ and R¹³⁰ are independently selected from the group consisting of: hydrogen and halogen; R¹²⁹ is selected from the group consisting of: (i) hydrogen; (ii) halogen; (iii) alkoxy —OR¹⁰ where R¹⁰ is selected from the group consisting of: (a) a straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and (b) a C-1 to C-6 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; and (iv) straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R¹³² is selected from the group consisting of: hydrogen and straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and Formula (32) excludes a compound of Formula (31):

or a stereoisomer or pharmaceutically acceptable salt thereof; wherein n is 1 or 2; R¹³³ and R¹³⁷ are independently selected from the group consisting of: hydrogen; halogen; and straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R¹³⁴ and R¹³⁶ are independently selected from the group consisting of: hydrogen; trifluoromethyl; and halogen; R¹³⁵ selected from the group consisting of: hydrogen and halogen; R¹³⁸, R¹⁴⁰, and R¹⁴¹ are hydrogen; R¹³⁹ is selected from the group consisting of: hydrogen and halogen; R¹⁴² is selected from the group consisting of: hydrogen and straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; and Formula (33) excludes a compound of Formula (31):


11. The compound of claim 10, wherein (A) the compound is according to Formula (32) (11091-like Type H) and one or more of the following features are satisfied: R¹²² and R¹²⁶ are independently selected from the group consisting of hydrogen; trifluoromethyl; methoxy; and chloro; R¹²² or R¹²⁶ is hydrogen; R¹²³ and R¹²⁵ are independently selected from the group consisting of halogen and trifluoromethyl; R¹²⁴ selected from the group consisting of halogen and straight chain or branched C-1 to C-4 lower alkyl optionally containing unsaturation; R¹²⁷ and R¹³¹ are independently selected from the group consisting of hydrogen, methyl, and methoxy; R¹²⁸ and R¹³⁰ are independently selected from the group consisting of hydrogen and chloro; R¹²⁹ is selected from the group consisting of hydrogen; chloro; methoxy; and methyl. R¹³² of Formula (32) is selected from the group consisting of hydrogen; methyl; and ethyl; or the compound is selected from the group consisting of:

or (B) the compound is according to Formula (33) (11091-like Type I) and one or more of the following features are satisfied: R¹³³ and R¹³⁷ are independently selected from the group consisting of: hydrogen; methyl; and chloro. R¹³⁴ and R¹³⁶ are independently selected from the group consisting of: hydrogen; trifluoromethyl; and chloro. R¹³⁵ is chloro. R¹³⁹ is selected from the group consisting of: fluoro and hydrogen. R¹⁴² is selected from the group consisting of: hydrogen or methyl and the compound is selected from the group consisting of


12. The compound of claim 10 that inhibits EGFR activity comprising: at least nine or more of functional groups F(V)1, F(V)2, F(V)3, F(V)4, F(V)5, F(V)6, F(V)7, F(V)8, F(V)9, F(V)10, and F(V)11 of a Scheme V 11091-like pharmacophore; wherein functional group F(V)1 accepts a hydrogen bond from a side chain amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=85.329, θ (theta)=34.962, and φ (phi)=53.394 and a spherical radius of about 1.2 Å; functional group F(V)2 accepts a hydrogen bond from a side chain amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=87.180, θ (theta)=35.618, and φ (phi)=53.267 and a spherical radius of about 1.5 Å; functional group F(V)3 accepts a hydrogen bond from a side chain amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=86.349, θ (theta)=34.560, and φ (phi)=55.424 and a spherical radius of about 1.5 Å; functional group F(V)4 donates a hydrogen bond to a side chain carbonyl of receptor Gln411 of SEQ ID NO: 1 and has coordinates of r=83.958, θ (theta)=35.296, and φ (phi)=51.296 and a spherical radius of about 1.2 Å; functional group F(V)5 donates a hydrogen bond to a side chain carbonyl of receptor Gln411 of SEQ ID NO: 1 and has coordinates of r=82.060, θ (theta)=34.984, and φ (phi)=50.365 and a spherical radius of about 1.5 Å; functional group F(V)6 donates a hydrogen bond to a side chain nitrogen of receptor Gln411 of SEQ ID NO: 1 and has coordinates of r=83.884, θ (theta)=36.166, and φ (phi)=49.227 and a spherical radius of about 1.2 Å; functional group F(V)7 donates a hydrogen bond to a side chain nitrogen of receptor Gln411 of SEQ ID NO: 1 and has coordinates of r=82.006, θ (theta)=35.523, and φ (phi)=49.076 and a spherical radius of about 1.5 Å; functional group F(V)8 accepts a hydrogen bond to a side chain imidazole of receptor His409 of SEQ ID NO: 1 and has coordinates of r=85.100, θ (theta)=38.590, and φ (phi)=43.143 and a spherical radius of about 1.2 Å; functional group F(V)9 forms a favorable π-π interaction with a phenyl ring of receptor Phe412 of SEQ ID NO: 1 and has coordinates of r=84.418, θ (theta)=37.489, and φ (phi)=46.968 and a spherical radius of about 1.2 Å; functional group F(V)10 forms a favorable π-π interaction with a phenyl ring of receptor Phe412 of SEQ ID NO: 1 and has coordinates of r=82.486, θ (theta)=38.027, and φ (phi)=47.246 and a spherical radius of about 1.5 Å; and functional group F(V)11 forms a favorable hydrophobic interaction with a side chain of receptors Val417 and Ile448 of SEQ ID NO: 1 and has coordinates of r=84.438, θ (theta)=40.753, and φ (phi)=48.557 and a spherical radius of about 1.2 Å; functional group F11 forms a favorable hydrophobic interaction with a side chain of receptors Val417 and Ile448 of SEQ ID NO: 1 and has coordinates of r=84.438, θ (theta)=40.753, and φ (phi)=48.557 and a spherical radius of about 1.2 Å; and the compound substantially maintains a non-extended tether inactive configuration of EGFR or substantially prevents stabilization of an extended tether active configuration of EGFR.
 13. A method of treating a proliferative disease, disorder, or condition, comprising: administering to a subject in need thereof a composition comprising a therapeutically effective amount of (i) a compound of any one of claims 1, 4, 7, or 10, or a stereoisomer or pharmaceutically acceptable salt thereof; (ii) a compound selected from the group consisting of:

or a stereoisomer or pharmaceutically acceptable salt thereof; or (iii) a compound selected from the group consisting of:

or a stereoisomer or pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier or excipient.
 14. The method of claim 13, wherein the proliferative disease, disorder, or condition is (i) selected from the group consisting of: cancer; a blood vessel proliferative disorder; a fibrotic disorder; a mesangial cell proliferative disorder; psoriasis; actinic keratoses; seborrheic keratoses; warts; keloid scars; eczema; and hyperproliferative diseases caused by a viral infection; (ii) is a disease, disorder, or condition associated with EGFR; or (iii) is a disease, disorder, or condition associated with EGFR selected from the group consisting of: cancer; a blood vessel proliferative disorder; a fibrotic disorder; a mesangial cell proliferative disorder; psoriasis; actinic keratoses; seborrheic keratoses; warts; keloid scars; eczema; and hyperproliferative diseases caused by a viral infection.
 15. A method for identifying an epidermal growth factor receptor (EGFR) inhibitor, comprising: providing a pharmacophore comprising a scheme selected from the group consisting of: Scheme II, Scheme III, Scheme IV, or Scheme V as input to a 3-dimensional database; comparing a three dimensional structure of a candidate compound to the three dimensional structure of the pharmacophore; selecting a candidate compound with a three dimensional structure that substantially aligns with: five or more functional groups of Scheme II (ADS-1734-like); seven or more functional groups of Scheme III (AD4-1886-like); eight or more of functional groups of Scheme IV (AD4-10381-like); or nine or more of functional groups of Scheme V (AD4-11091-like); wherein similarity between the three-dimensional structure of the candidate compound and the three-dimensional structure of the pharmacophore is indicative of an ability of the candidate compound to inhibit EGFR by substantially maintaining a tethered inactive configuration of EGFR or substantially preventing stabilization of the untethered active configuration of EGFR; Scheme II (ADS-1734-like) comprises functional groups F(II)1, F(II)2, F(II)3, F(II)4, F(II)5, F(II)6, F(II)7, F(II)8, and F(II)9; wherein functional group F(II)1 donates an H-bond or forms a salt bridge to a carboxylate side chain of receptor Asp553 of SEQ ID NO: 1 and has coordinates of r=56.363, θ (theta)=94.368, and φ (phi)=−17.752 and a spherical radius of about 1.2 Å; functional group F(II)2 is a donor and has coordinates of r=53.290, θ (theta)=101.494, and φ (phi)=−23.244 and a spherical radius of about 1.0 Å; functional group F(II)3 forms a hydrophobic contact with a side chain of receptor Val568, imidazolidine side chain of receptor Pro552 and with a side chain of Met253 of SEQ ID NO: 1 and has coordinates of r=53.726, θ (theta)=97.830, and φ (phi)=−18.377 and a spherical radius of about 1.7 Å; functional group F(II)4 forms a hydrophobic contact with side chain of receptor Val575, Met253, and with an imidazolidine ring of receptor Pro552 of SEQ ID NO: 1 and has coordinates of r=53.647, θ (theta)=103.844, and φ (phi)=−20.990 and a spherical radius of about 1.4 Å; functional group F(II)5 donates an H-bond to a side chain hydroxyl of Thr570 of SEQ ID NO: 1 and has coordinates of r=51.093, θ (theta)=104.261, and φ (phi)=−25.552 and a spherical radius of about 1.2 Å; functional group F(II)6 is a donor having directionality of F4 with respect to a backbone carbonyl of receptor Thr570 of SEQ ID NO: 1 and has coordinates of r=52.340, θ (theta)=103.980, and φ (phi)=−27.461 and a spherical radius of about 1.5 Å; functional group F(II)7 accepts an H-bond from a receptor backbone NH of Ala573 of SEQ ID NO: 1 and has coordinates of r=51.383, θ (theta)=106.455, and φ (phi)=−24.319 and a spherical radius of about 1.2 Å; functional group F(II)8 is an acceptor having directionality of F7 with respect to the backbone NH of receptor Ala573 of SEQ ID NO: 1 and has coordinates of r=52.861, θ (theta)=107.691, and φ (phi)=−25.448 and a spherical radius of about 1.5 Å; and functional group F(II)9 donates an H-bond to a backbone carbonyl of Asp563 and forms a salt bridge to a side chain carboxylate of receptor Asp563 of SEQ ID NO: 1 and has coordinates of r=57.688, θ (theta)=99.198, and φ (phi)=−21.588 and a spherical radius of about 1.2 Å; Scheme III (AD4-1886-like) comprises functional groups F(III)1, F(III)2, F(III)3, F(III)4, F(III)5, F(III)6, F(III)7, F(III)8, and F(III)9; wherein functional group F(III)1 donates a hydrogen bond to a side chain carbonyl of receptor Gln408 and accepts a hydrogen bond from the side chain NH₂ of receptor Gln384 of SEQ ID NO: 1 and has coordinates of r=81.552, θ (theta)=41.243, and φ (phi)=45.369 and a spherical radius of about 1.2 Å; functional group F(III)2 accepts a hydrogen bond from a side chain OH of receptor Ser440 of SEQ ID NO: 1 and has coordinates of r=87.287, θ (theta)=40.739, and φ (phi)=51.781 and a spherical radius of about 1.2 Å; functional group F(III)3 accepts a hydrogen bond from a side chain OH of receptor Ser440 of SEQ ID NO: 1 and has coordinates of r=86.320, θ (theta)=41.915, and φ (phi)=52.323 and a spherical radius of about 1.5 Å; functional group F(III)4 forms a favorable coulombic interaction with an imidazole side chain of receptor His409 of SEQ ID NO: 1 and has coordinates of r=85.870, θ (theta)=38.463, and φ (phi)=41.650 and a spherical radius of about 1.2 Å; functional group F(III)5 forms a favorable coulombic interaction with an imidazole side chain of receptor His409 of SEQ ID NO: 1 and has coordinates of r=82.241, θ (theta)=37.431, and φ (phi)=44.151 and a spherical radius of about 1.5 Å; functional group F(III)6 accepts a hydrogen bond from, or forms a salt bridge to, NH³⁺ of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=88.009, θ (theta)=37.822, and φ (phi)=54.903 and a spherical radius of about 1.2 Å; functional group F(III)7 accepts a hydrogen bond from, or forms a salt bridge to, NH³⁺ of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=86.513, θ (theta)=36.889, and φ (phi)=54.484 and a spherical radius of about 1.5 Å; functional group F(III)8 donates a hydrogen bond to a side chain carbonyl of receptor Gln408 of SEQ ID NO: 1 and has coordinates of r=81.552, θ (theta)=41.243, and φ (phi)=45.369 and a spherical radius of about 1.2 Å; and functional group F(III)9 donates a hydrogen bond to a side chain carbonyl of receptor Gln408 of SEQ ID NO: 1 and has coordinates of r=79.652, θ (theta)=40.928, and φ (phi)=44.528 and a spherical radius of about 1.6 Å; Scheme IV (AD4-10381-like) comprises functional group F(IV)1, F(IV)2, F(IV)3, F(IV)4, F(IV)5, F(IV)6, F(IV)7, F(IV)8, and F(IV)9; wherein functional group F(IV)1 accepts a hydrogen bond from receptor side chain OH of Thr239 of SEQ ID NO: 1 and has coordinates of r=49.686, θ (theta)=113.993, and φ (phi)=−17.014 and a spherical radius of about 1.2 Å; functional group F(IV)2 accepts a hydrogen bond from receptor side chain OH of Thr239 of SEQ ID NO: 1 and has coordinates of r=48.071, θ (theta)=115.388, and φ (phi)=−16.211 and a spherical radius of about 1.6 Å; functional group F(IV)3 forms a hydrophobic contact with a side chain of receptor Met576 and imidazolidine ring of Pro242 of SEQ ID NO: 1 and has coordinates of r=50.781, θ (theta)=113.121, and φ (phi)=−17.520 and a spherical radius of about 1.2 Å; functional group F(IV)4 forms a hydrophobic contact with a side chain of receptor Met576 and imidazolidine ring of Pro242 of SEQ ID NO: 1 and has coordinates of r=52.021, θ (theta)=114.264, and φ (phi)=−15.878 and a spherical radius of about 1.5 Å; functional group F(IV)5 forms a hydrophobic contact with a side chain of receptor Met576 and imidazolidine ring of Pro242 of SEQ ID NO: 1 and has coordinates of r=50.322, θ (theta)=111.253, and φ (phi)=−15.426 and a spherical radius of about 1.2 Å; functional group F(IV)6 forms a hydrophobic contact with a side chain of receptor Met576 and imidazolidine ring of Pro242 of SEQ ID NO: 1 and has coordinates of r=51.575, θ (theta)=112.433, and φ (phi)=−13.827 and a spherical radius of about 1.5 Å; functional group F(IV)7 forms a hydrophobic contact with a side chain of receptor Leu243 and a side chain of Thr239 of SEQ ID NO: 1 and has coordinates of r=47.767, θ (theta)=112.521, and φ (phi)=−10.196 and a spherical radius of about 1.2 Å; functional group F(IV)8 donates a hydrogen bond to a backbone carbonyl of His280 of SEQ ID NO: 1 and has coordinates of r=45.184, θ (theta)=112.328, and φ (phi)=−8.875 and a spherical radius of about 1.2 Å; functional group F(IV)9 forms a hydrophobic contact with a side chain of receptor Met244 and Leu243 of SEQ ID NO: 1 and has coordinates of r=49.512, θ (theta)=108.007, and φ (phi)=−8.504 and a spherical radius of about 1.2 Å; and functional group F(IV)10 forms a hydrophobic contact with a side chain of receptor Met244 of SEQ ID NO: 1 and has coordinates of r=50.431, θ (theta)=104.985, and φ (phi)=−7.420 and a spherical radius of about 1.8 Å; and Scheme V (AD4-11091-like) comprises functional group functional groups F(V)1, F(V)2, F(V)3, F(V)4, F(V)5, F(V)6, F(V)7, F(V)8, F(V)9, F(V)10, and F(V)11; wherein functional group F(V)1 accepts a hydrogen bond from a side chain amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=85.329, θ (theta)=34.962, and φ (phi)=53.394 and a spherical radius of about 1.2 Å; functional group F(V)2 accepts a hydrogen bond from a side chain amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=87.180, θ (theta)=35.618, and φ (phi)=53.267 and a spherical radius of about 1.5 Å; functional group F(V)3 accepts a hydrogen bond from a side chain amino of receptor Lys465 of SEQ ID NO: 1 and has coordinates of r=86.349, θ (theta)=34.560, and φ (phi)=55.424 and a spherical radius of about 1.5 Å; functional group F(V)4 donates a hydrogen bond to a side chain carbonyl of receptor Gln411 of SEQ ID NO: 1 and has coordinates of r=83.958, θ (theta)=35.296, and φ (phi)=51.296 and a spherical radius of about 1.2 Å; functional group F(V)5 donates a hydrogen bond to a side chain carbonyl of receptor Gln411 of SEQ ID NO: 1 and has coordinates of r=82.060, θ (theta)=34.984, and φ (phi)=50.365 and a spherical radius of about 1.5 Å; functional group F(V)6 donates a hydrogen bond to a side chain nitrogen of receptor Gln411 of SEQ ID NO: 1 and has coordinates of r=83.884, θ (theta)=36.166, and φ (phi)=49.227 and a spherical radius of about 1.2 Å; functional group F(V)7 donates a hydrogen bond to a side chain nitrogen of receptor Gln411 of SEQ ID NO: 1 and has coordinates of r=82.006, θ (theta)=35.523, and φ (phi)=49.076 and a spherical radius of about 1.5 Å; functional group F(V)8 accepts a hydrogen bond to a side chain imidazole of receptor His409 of SEQ ID NO: 1 and has coordinates of r=85.100, θ (theta)=38.590, and φ (phi)=43.143 and a spherical radius of about 1.2 Å; functional group F(V)9 forms a favorable π-π interaction with a phenyl ring of receptor Phe412 of SEQ ID NO: 1 and has coordinates of r=84.418, θ (theta)=37.489, and φ (phi)=46.968 and a spherical radius of about 1.2 Å; functional group F(V)10 forms a favorable π-π interaction with a phenyl ring of receptor Phe412 of SEQ ID NO: 1 and has coordinates of r=82.486, θ (theta)=38.027, and φ (phi)=47.246 and a spherical radius of about 1.5 Å; and functional group F(V)11 forms a favorable hydrophobic interaction with a side chain of receptors Val417 and Ile448 of SEQ ID NO: 1 and has coordinates of r=84.438, θ (theta)=40.753, and φ (phi)=48.557 and a spherical radius of about 1.2 Å.
 16. The method of claim 15, further comprising one or more of the following features: determining identity and spatial orientation of at least a portion of atoms of EGFR associated with stabilizing a tethered configuration of domain II and domain IV of EGFR in an inactive conformation; and constructing a pharmacophore, wherein the pharmacophore comprises a pluralitiy of pharmacophoric features that approximates the identity and the spatial orientation of the at least a portion of atoms of EGFR associated with stabilizing a tethered configuration of domain II and domain IV of EGFR in an inactive conformation such that the pharmacophore structural features are complementary to the inactive EGFR configuration. determining identity and spatial orientation of at least a portion of atoms of EGFR associated with stabilizing a tethered configuration of domain II and domain IV of EGFR in an inactive conformation by analysis of X-ray crystallographic data derived from a crystalline form of EGFR in an inactive, tethered conformation; wherein at least one pharmacophoric feature approximates identity and spatial orientations of at least a portion of atoms of domain II of EGFR in a tethered inactive conformation; wherein at least one pharmacophoric feature approximates identity and spatial orientations of at least a portion of atoms of a cleft region between domain II and domain IV of EGFR in a tethered inactive conformation; or determining a docking affinity of the candidate molecule for the at least a portion of atoms of EGFR associated with stabilizing a tethered configuration of domain II and domain IV of EGFR in an inactive conformation, wherein docking affinity is quantified by energy gained upon interaction of the candidate molecule with the target biomolecule, energy required to attain the docked conformation relative to the lowest energy conformation, or a combination thereof. 